2-oxy-4H-3,1-benzoxazin-4-ones and pharmaceutical use

ABSTRACT

2-Oxy-4H-3,1-benzoxazin-4-ones represented by the formula: ##STR1## and the pharmaceutically acceptable acid addition salts thereof, wherein: a is an integer of 0-4; 
     A is a bond, or alkylene having one to eight carbon atoms; 
     R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to six carbon atoms, wherein the phenyl, imidazolyl or cycloalkyl ring is optionally substituted with 1-3 substituents independently selected from the group consisting of lower alkyl having one to four carbon atoms, lower alkoxy having one to four carbon atoms, --N(R 1 ) 2 , --NO 2 , halo or lower alkylthio having one to four carbon atoms, and, 
     each R&#39; is independently selected from the group consisting of lower alkyl having one to six atoms, lower alkenyl having two to six carbon atoms, lower alkoxy having one to six carbon atoms, 
     lower alkylthio or halo-lower alkyl having one to four carbon atoms, halo, --NO 2 , --N(R 1 ) 2 , ##STR2##  --NR 1  COR 2 , and ##STR3##  in which each R 1  is independently hydrogen or lower alkyl having one to four carbon atoms, or together form a piperidine or a piperazine ring optionally substituted at the ring nitrogen by lower alkyl having one to four carbon atoms or --CH 2  CH 2  OH, 
     each R 2  is independently lower alkyl having one to four carbon atoms, 
     A is an alkylene group if R is hydrogen, 
     and the pharmaceutically acceptable acid addition salts thereof are useful as serine protease inhibitors in humans and animals.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to (i) 2-oxy-4H-3,1-benzoxazin-4-ones and thepharmaceutically acceptable acid addition salts thereof; (ii) the use ofthese compounds as serine protease inhibitors in humans and animals;(iii) pharmaceutical compositions comprising a compound of thisinvention and at least one pharmaceutical excipient; and (iv) processesfor preparing the compounds of this invention.

2. Related Art

The compounds of this invention are 2-oxy substituted derivatives of4H-3,1-benzoxazinones having the following structure: ##STR4##

2-Ethoxy-4H-3,1-benzoxazin-4-one is disclosed in British PatentSpecification No. 1,389,128 and in the corresponding GermanOffenlegungschrift No. 2241012. A few 4H-3,1-benzoxazin-4-ones are knownto possess enzyme-inhibitory activity. Teshima et al. have disclosedvarious 2-alkyl-4H-3,1-benzoxazin-4-ones reported to be active as enzymeinhibitors. (J. Biol. Chem., 257, 5085-5091, 1982).4H-3,1-benzoxazin-2,4-dione has been disclosed as having some enzymeinhibitory activity (Moorman, A. R., and Abeles, R. H. J. Amer. Chem.Soc. 104, 6785-6786, 1982). 2-Ethoxy-4H-3,1-benzoxazinone and2-(trifluoromethyl)-4H-3,1-benzoxazinone are believed to inhibitchymotrypsin (Hedstrom et al, Biochemistry 23, 1753-1759, 1984).

SUMMARY

We have discovered that the class of 2-oxy-4H-3,1-benzoxazin-4-ones ofFormula I, as shown below: ##STR5## and described and claimed herein,are biologically active as inhibitors of enzymes, and in particular areinhibitors of serine proteases. Within this class, several groups ofcompounds are novel and preferred. Accordingly, the invention describedand claimed herein contains the following aspects:

1. Novel compounds of Formula I wherein:

a is an integer of 0-4;

A is a bond, or alkylene having one to eight carbon atoms;

R is phenyl, imidazolyl, or cycloalkyl having three to six carbon atoms,wherein the phenyl or cycloalkyl ring is optionally substituted with 1-3substituents independently selected from the group consisting of loweralkyl having one to four carbon atoms, lower alkoxy having one to fourcarbon atoms, --N(R¹)₂, --NO₂, halo, and lower alkylthio having one tofour carbon atoms, and

each R' is independently selected from the group consisting of loweralkyl having one to six atoms, lower alkenyl having two to six carbonatoms, lower alkoxy having one to six carbon atoms, --NO₂, halo-loweralkyl or lower alkylthio having one to six carbon atoms, halo, --N(R¹)₂,##STR6## --NR¹ COR² and ##STR7## in which each R¹ is independentlyhydrogen or lower alkyl having one to four carbon atoms, or togetherform a piperidine or piperazine ring optionally substituted at the ringnitrogen by lower alkyl having one to four carbon atoms or --CH₂ CH₂ OH,and

each R² is independently lower alkyl having one to four carbon atoms,

and the pharmaceutically acceptable acid addition salt thereof.

2. Novel compounds of Formula I wherein:

a is an integer of 1-4;

A is alkylene having one to eight carbon atoms;

R is hydrogen; and

each R' is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, halo-lower alkyl or lower alkylthio having one to sixcarbon atoms, halo, --NO₂, --N(R¹)₂, ##STR8## --NR¹ COR², and in which

each R¹ is independently hydrogen or lower alkyl having one to fourcarbon atoms, or together form a piperidine or piperazine ringoptionally substituted at the ring nitrogen by lower alkyl having one tofour carbon atoms or --CH₂ CH₂ OH, and

each R² is independently lower alkyl having one to four carbon atoms,

or a pharmaceutically acceptable acid addition salt thereof.

3. Novel compounds of Formula I wherein:

a is an integer of 0-4;

A is alkylene having four to eight carbon atoms;

R is hydrogen; and

each R' is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, halo, lower alkoxy having one to six carbon atoms, loweralkylthio or halo-lower alkyl having one to six carbon atoms, --NO₂,--N(R¹)₂, ##STR9## --NR¹ COR², and ##STR10## in which each R¹ isindependently hydrogen or lower alkyl having one to six carbon atoms, ortogether form a piperidine or piperazine ring optionally substituted atthe ring nitrogen by lower alkyl having one to four carbon atoms or--CH₂ CH₂ OH, and

each R² is independently lower alkyl having one to four carbon atoms,

or a pharmaceutically acceptable acid addition salt thereof.

4. Novel compounds of Formula A: ##STR11## wherein: a is an integer of0-3;

A is a bond, or alkylene having one to eight carbon atoms;

R is hydrogen, imidazolyl, phenyl, or cycloalkyl having three to sixcarbon atoms, wherein the phenyl, imidazolyl or cycloalkyl ring isoptionally substituted with 1-3 substituents independently selected fromthe group consisting of lower alkyl having one to four carbon atoms,lower alkoxy having one to four carbon atoms, --N(R¹)₂, --NO₂, halo, andlower alkylthio having one to four carbon atoms; and

each R' and R" are independently selected from the group consisting oflower alkyl having one to six carbon atoms, lower alkenyl having two tosix carbon atoms, lower alkoxy having one to six carbon atoms, loweralkylthio or halo-lower alkyl having one to six carbon atoms, --NO₂,--N(R¹)₂, --NR¹ COR², ##STR12## in which each R¹ is independentlyhydrogen or lower alkyl having one to four carbon atoms, or togetherform a piperidine or piperazine ring optionally substituted at the ringnitrogen with lower alkyl having one to four carbon atoms or --CH₂ CH₂OH;

each R² is independently lower alkyl having one to four carbon atoms,and

A is alkylene if R is hydrogen,

or a pharmaceutically acceptable acid addition salt thereof.

5. Pharmaceutical compositions which comprise a therapeuticallyeffective amount of a compound of Formula I or A chosen from among thosedescribed in Groups 1-4 above, or a pharmaceutically acceptable acidaddition salt thereof, in admixture with at least one pharmaceuticallyacceptable excipient.

6. Methods of inhibiting serine proteases in humans and animals whichcomprise administering to a subject in need of such treatment atherapeutically effective amount of a compound of Formula I or A chosenfrom among those described in Groups 1 to 4, above.

7. A method of inhibiting serine proteases in humans and animals whichmethod comprises administering to a subject in need of such treatment atherapeutically effective amount of a compound of the formula: ##STR13##wherein: a is an integer of 0-4;

A is a bond, or alkylene having one to eight carbon atoms;

R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to sixcarbon atoms, wherein the phenyl or cycloalkyl ring is optionallysubstituted with 1-3 substituents independently selected from the groupconsisting of lower alkyl having one to four carbon atoms, lower alkoxyhaving one to four carbon atoms, --N(R¹)₂, --NO₂, halo, and lowerthioalkyl having one to four carbon atoms; and

each R' is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, halo, lower alkoxy having one to six carbon atoms, loweralkylthio or halo-lower alkyl having one to six carbon atoms, --N(R¹)₂,##STR14## --NR¹ COR², and ##STR15## in which each R¹ is independentlyhydrogen or lower alkyl having one to four carbon atoms, or togetherform a piperidine or piperazine ring optionally substituted at the ringnitrogen with lower alkyl having one to four carbon atoms or --CH₂ CH₂OH;

each R² is independently lower alkyl having one to four carbon atoms,and

A is alkylene if R is hydrogen,

or a pharmaceutically acceptable acid addition salt thereof.

8. Novel compounds of Formulas I and A useful as intermediates in thesynthesis of compounds of the invention, having the formula: ##STR16##wherein: a is an integer of 0-3;

A is a bond, or alkylene having one to eight carbon atoms;

R is hydrogen, phenyl, imidazolyl or cycloalkyl having three to sixcarbon atoms, wherein the phenyl or cycloalkyl ring is optionallysubstituted with 1-3 substituents independently selected from the groupconsisting of lower alkyl having one to four carbon atoms, lower alkoxyhaving one to four carbon atoms, --N(R¹)₂, --NO₂, halo, and lowerthioalkyl having one to four carbon atoms;

R"' is lower alkyl having one to six carbon atoms; and

each R' is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, lower alkoxy having one to six carbon atoms, halo-loweralkyl or lower alkylthio having one to six carbon atoms, halo, --N(R¹)₂,##STR17## --NR¹ COR², and ##STR18## in which each R¹ is independentlyhydrogen or lower alkyl having one to four carbon atoms, or togetherform a piperidine or piperazine ring optionally substituted at the ringnitrogen with lower alkyl having one to four carbon atoms or --CH₂ CH₂OH;

each R² is independently lower alkyl having one to four carbon atoms,and

A is alkylene if R is hydrogen.

The foregoing paragraphs numbered 1-8 set forth aspects of the inventionwhich are referred to here and subsequently as "Groups" 1 to 8.

DETAILED DESCRIPTION OF THE INVENTION

As used herein:

"Alkylene" means a branched or unbranched saturated hydrocarbon bridginggroup having one to eight carbon atoms, including but not limited to,methylene, ethylene, propylene, isopropylene, n-propylene, butylene,sec-butylene, isobutylene, n-pentylene, hexylene, octylene, and thelike.

"Lower alkyl" means a branched or unbranched saturated hydrocarbon chainhaving, unless otherwise noted, one to six carbon atoms, including butnot limited to methyl, ethyl, propyl, isopropyl, n-propyl, butyl,sec-butyl, isobutyl, n-pentyl, hexyl, octyl and the like. Lower alkylgroups may be limited to fewer than six carbon atoms when specificallydesignated, e.g. "R² is lower alkyl having one to four carbon atoms."

"Lower alkenyl" means a branched or unbranched unsaturated hydrocarbonchain of 2 to 6 carbon atoms, including but not limited to vinyl, allyl,1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, isobutenyl,1,3-butadienyl, 1-pentenyl, 2-pentenyl, isoprenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, cis-2-butenyl, trans-2-butenyl, cis-2-pentenyl,trans-2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl and2,3-dimethyl-2-butenyl.

"Lower alkoxy" means the group --OR wherein R is lower alkyl as hereindefined.

"Lower alkylthio" means the group --SR wherein R is lower alkyl asherein defined.

"Halo" refers to chloro, bromo and iodo.

"Halo-lower alkyl" means the group --R-halo in which R is lower alkyl,and both lower alkyl and halo have the definitions given herein. Thealkyl group may bear one or two halo substituents; examples include butare not limited to bromomethyl, dibromomethyl, chloroethyl,dichloroethyl, and the like.

"Pharmaceutically acceptable acid addition salt" refers to those saltswhich retain the biological effectiveness and properties of the freebases and which are not biologically or otherwise undesirable, formedwith inorganic acids such as hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid and the like, and organicacids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,oxalic acid, malic acid, malonic acid, succinic acid, maleic acid,fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid,mandelic acid, methanesulfonic acid, ethane sulfonic acid,p-toluenesulfonic acid, ascorbic acid, salicylic acid and the like.

"Optional" or "optionally" means that the subsequently described eventor circumstances may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances in whichit does not. For example, "phenyl . . . optionally substituted" meansthat the phenyl may or may not be substituted and that the descriptionincludes both unsubstituted phenyl and phenyl wherein there issubstitution.

Certain of the compounds of the invention have chiral centers and existas optical antipodes. The invention described and claimed hereinincludes each of the individual enantiomers as well as their racemicmodifications and the racemic mixture.

The compounds of this invention are named as2-oxy-4H-3,1-benzoxazin-4-ones using the numbering system set forthbelow. ##STR19##

For example, the compound of Formula I where AR is isopropyl, a is 1 andR' is methyl in the 5-position is named2-isopropyloxy-5-methyl-4H-3,1-benzoxazin-4-one.

The compound where AR is phenethyl, a is 2 and both R's are amino in the5- and 7-positions is named2-(phenethyloxy)-5,7-di-amino-4H-3,1-benzoxazin-4-one.

The compound of Formula I where AR is cyclohexyl, and a is zero is named2-cyclohexyloxy-4H-3,1-benzoxazin-4-one.

The compound of Formula I where AR is n-butyl, a is 1 and R' ismethylthio in the 5-position is named2-n-butoxy-5-methylthio-4H-3,1-benzoxazin-4-one.

Preferred Embodiments

Within the several aspects of this invention which are set fourth asGroups 1 to 4 in the Summary of the Invention, certain subgroups arepreferred. The metes and bounds of these subgroups and their relativedegrees of preference are described below.

Within each of the groups of compounds defined as Groups 1 to 4,preferred subgroups are compounds of Formulas I and A in which a is atleast one. Among these, preferred classes encompass compounds in whichthe R's are in the 5- and/or 7-positions. Within these classes,preferred subclasses include compounds of Formula I in which an R' (orR" in group 4) is in the 5-position; of these, especially preferred arecompounds of Formula I in which an R' is also in 7-position.

Preferred R' or R" substituents at the 5-position are lower alkyl havingone to six carbon atoms and lower alkenyl having two to six carbonatoms. Especially preferred R' and R" substituents at the 5-position arelower alkyl having one to six carbon atoms, particularly one to threecarbon atoms, and most particularly methyl or ethyl. Preferred R'substituents at the 7-position are lower alkoxy having one to six carbonatoms, --N(R¹)₂, ##STR20## --NR¹ COR² and ##STR21## particularly--N(R¹)₂ and --NR¹ COOR², especially where each R' is independentlyhydrogen, methyl or ethyl, and R² is methyl or ethyl.

Additionally, certain subgroups within each individual Group havepreference. Among the compounds of Formula I as set forth in Group 1, apreferred subgroup is those compounds in which A is a bond, or alkylenehaving one to four carbon atoms, especially a bond or methylene. Amongthe compounds of Formula I defined by Group 2, a preferred subgroup arethose compounds in which A is lower alkylene having one to four carbonatoms, particularly methylene and ethylene. Among the compounds ofFormula I as set forth in Group 3, a preferred subgroup encompassescompounds in which A is alkylene having five to eight carbon atoms.Among the compounds of Formula A as defined in Group 4, a preferredsubgroup consists of compounds in which a is zero or one and A is loweralkylene having one to four carbon atoms, particularly methylene andethylene.

At the present time, the most preferred compounds of this invention are:

2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one;

7-amino-2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one, and

7-amino-2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one.

Methods of Preparation

In this section the remainder of the specification, the compounds of theinvention which are referred to in the Summary of the Invention and inthe Claims as compounds of Formula I and compounds of Formula A will bereferred to collectively as compounds of Formula I, and further definedas compounds of Formula IA, IB, IC, etc.

A. Compounds of Formula I in Which R' is Lower Alkyl, Lower Alkoxy,Lower Alkylthio, Halo, --NO₂ or an Amine Other than NH₂ (Compounds ofFormula IA).

The compounds of Formula I in which each R' substituent is lower alkyl,lower alkoxy, lower alkylthio, halo, --NO₂ or an amine other than --NH₂,(hereinafter defined as compounds of Formula IA), can be prepared by thegeneral procedure set forth in Reaction Scheme I, below. ##STR22##

As outlined in Reaction Scheme I, the compounds of Formula IA areprepared by cyclization of the corresponding appropriately substitutedor unsubstituted anthranilic acid. Cyclization is preferably achieved byreaction of the chosen anthranilic acid with about 3 to about 5,preferably about 4 equivalents of the desired phenyl-, cycloalkyl- oralkyl chloroformates of the formula ClCOOAR, wherein A and R are definedas hereinabove. The reaction takes place in a basic organic solvent suchas triethylamine, or, preferably, pyridine, and is carried out at roomtemperature over a period of about 0.5 to about 5 hours, preferablyabout 1 to about 3 hours. The final product, a compound of Formula IA,is then isolated by conventional means.

Isolation and purification of the final compounds and intermediatesdescribed herein can be effected, if desired, by any suitable separationor purification procedure such as, for example, filtration, extraction,crystallization, column chromatography, thin-layer chromatography, thicklayer chromatography, or a combination of these procedures. Specificillustrations of suitable separation and isolation procedures can be hadby reference to the examples hereinbelow. However, other equivalentseparation or isolation procedures could, of course, also be used.

Unsubstituted anthranilic acid is readily commercially available. Thesubstituted anthranilic acids (Formula II) used in preparing thecompounds of this invention are either commercially available, or can beprepared by methods well known in the art. The commercially availableanthranilic acids include, but are not limited to, 3-methyl-anthranilicacid, 4-methyl anthranilic acid, 5-methyl-anthranilic acid,6-methyl-anthranilic acid, 5-iodo-anthranilic acid, 4-nitro-anthranilicacid, and 4,5-dimethoxy-anthranilic acid. A list of commerciallyavailable anthranilic acids is available in Chem. Sources-U.S.A., 24thEd., 1983, Directories Publishing Company, Inc., Ormond Beach, Fla.Appropriately substituted anthranilic acids which are not commerciallyavailable can be readily prepared by methods known in the art. Suitablemethods include those of B. R. Baker, et al., J. Org. Chem., 17, 141,(1952) and of L. A. Paquette, et al., J. Am. Chem. Soc. 99, 3734,(1981). The former method involves the preparation of an isatin from asubstituted aniline derivative. Subsequent oxidation of the isatin givesthe anthranilic acid. The latter procedure employs the reduction of thecorresponding aromatic nitro-derivative to the anthranilic acid. Thesemethods are further illustrated in Preparation I, below.

The phenyl-, cycloalkyl- and alkyl chloroformates of Formula III areeither commercially available, or can be prepared by methods well knownor readily available in the chemical literature. Commercially availablechloroformates include, but are not limited to benzyl chloroformate,methyl chloroformate, ethyl chloroformate, n-butyl chloroformate,isobutyl chloroformate. Suitable chloroformates which are notcommercially available can be prepared by known methods. Preparativemethods include those of D. H. R. Barton et al., J. Chem Soc. 18 55-1857(1968) and K. Kurita et al., J. Org Chem. 41, 2070-2071, (1976). Theformer method involves treatment of an appropriate alcohol with phosgenein an inert solvent such as anhydrous ether. Further description of thismethod is provided in Preparation II, below. The latter procedureinvolves the reaction of an appropriate alcohol with trichloromethylchloroformate (diphosgene) in dry dioxane at refluxing temperature.

B. Compounds of Formula I in Which an R' is NH₂ (Compounds of FormulaIB)

Compounds of Formula I in which one of the R' substituents is NH₂(hereinafter referred to as compounds of Formula IB) can be prepared bythe procedure set forth in Reaction Scheme II, below. ##STR23##

As shown above, a nitro-substituted 2-oxy-4H-3,1-benzoxazin-4-one ofFormula IA is reduced to the corresponding amino-substituted compound ofFormula I by transfer hydrogenation. The reaction takes place in benzeneor THF in the presence of 10% Pd-C as catalyst and cyclohexene ashydrogen donor and is carried out at reflux temperature over about oneto five, preferably about three hours. The final product, a compound ofFormula IB, is then isolated by conventional means.

The nitro-substituted benzoxazinone of Formula IA may be commerciallyobtained, or is prepared according to the procedure set forth above inSection A.

C. Compounds in Which an R' is --NR¹ COR² (Compounds of Formula IC)

Compounds of the invention in which one of the R' substituents is anamine of the formula --NR¹ COR², (hereinafter referred to as compoundsof Formula IC) can be prepared from the corresponding amino-substitutedcompound of Formula IB, as shown in Reaction Scheme III. ##STR24##

The amino-substituted compound of Formula IA or IB, prepared asdescribed in section A or B, above, is reacted with about 1 to 3equivalents of an appropriate acid anhydride of Formula IV, with orwithout the presence of an inert organic solvent such as dichloromethaneor tetrahydrofuran. The reaction takes place at room temperature over aperiod of about 15 min to about 3 hrs, usually about 1 hour, and thefinal product, a compound of Formula IC, is then isolated byconventional means.

The acid anhydrides (Formula IV) used in preparing the compounds ofFormula IC are either commercially available or can be prepared bymethods well known in the art. The commercially available acidanhydrides include, but are not limited to, acetic anhydride, propionicanhydride, and butyric anyhydride. A list of commercially available acidanhydrides is available in Chem. Sources-U.S.A., 24th Ed., 1983,Directories Publishing Company, Inc., Ormond Beach, Fla. Acid anhydrideswhich are not commercially available can be readily prepared by methodsknown in the art. Suitable methods include the direct removal of waterfrom acids to form acid anhydride and from acid chlorides and alkalisalts of carboxylic acids by mixing the reactants and distilling off theanhydride formed as described in Preparative Organic Chemistry, editedby G. Hilgetag and A. Martini, page 387-390, John Wiley & Sons, NewYork-London-Sydney-Toronto (1972), and further illustrated inPreparation III, below.

D. Compounds in Which an R' is ##STR25## (Compounds of Formula ID)

Compounds of the invention which bear an R' substituent of the formula--NR¹ COOR² can be prepared by the procedure set forth in Section A,above, or from the corresponding amino-substituted compound of FormulaIA or IB, as shown below. ##STR26##

In carrying out the above conversion, the appropriate compound ofFormula IA or IB (prepared as described in Sections A and B, above)bearing an R¹ NH substituted as shown, is reacted with about one tothree equivalents, preferably about 1.5 equivalents, of an appropriatelychosen phenoxy-, cycloalkoxy- or alkoxy chloroformate of Formula III.(The commercial availability and methods of preparing the variouscompounds of Formula III used in making the compounds of this inventionare discussed in detail in Section A, hereinabove.) The reaction takesplace in an inert solvent such as dichloromethane or tetrahydrofuran inthe presence of a tertiary amine such as triethylamine or, preferably,pyridine, at room temperature over a period of about one to about three,usually about two, hours. The final product, a compound of Formula ID,is then isolated by conventional means.

E. Compounds in Which an R' is --NR¹ CON(R¹)₂ (Formula IE)

Compounds of the invention which bear an R' substituent of the formula--NR¹ CON(R¹)₂ (hereinafter referred to as compounds of Formula IE) canbe prepared from the corresponding amino-substituted compounds ofFormula IA and IB as shown below in Reaction Scheme V. ##STR27## Theamino-substituted compound of Formula IA or IB is reacted in an inertsolvent such as benzene or tetrahydrofuran with about 0.5 to 1preferably 0.75, equivalent of trichloromethyl chloroformate, for aperiod of about 15 to 60 minutes, preferably about 30 minutes. Withoutisolation, the resulting carbamyl chloride derivative is then treatedwith about 5 to 10, preferably about 7.5 equivalents of pyridine andabout 1 to 3, preferably about 2 equivalents of an appropriate primaryor secondary alkyl amine for a period of about 15 to 60, usually about30 minutes. The final product, a compound of Formula IE, is thenisolated by conventional means.

F. Compounds in which an R' is Halo-Lower Alkyl (Formula IF)

Compounds of the Formula I which bear an R' substituent in which R' islower alkyl can be mono- or di-halogenated at a benzylic position withan N-halo-succinimide such as N-bromosuccinimide, and AIBN(2-2'-azobis-isobutyronitrile) to give either the mono-halo compound ofFormula (IF₁) or the di-halo compound of Formula (IF₂). Normally, only acatalytic amount of AIBN is used. This procedure is shown n ReactionScheme VI, below, in which N-bromosuccinimide is used as an example.N-chlorosuccinimide or N-iodosuccinimide can also be used. ##STR28##

The halogenation reaction is carried out in carbon tetrachloride atreflux temperature for 2 to 4 hours, and the product is isolated byconventional means. When, for example, one equivalent ofN-bromosuccinimide is used, the monobromo compound of Formula IF₁ is themajor product; likewise, when two equivalents of N-bromosuccinimide areused, the dibromo compound of Formula IF₂ is the major product.

G. Compounds in which an R' is lower Alkenyl or Lower Alkyl

Compounds of Formula I in which an R' is lower alkenyl or lower alkylcan be prepared from the corresponding compounds of Formula IF₁ and IG₂,as shown in Reaction Scheme VII. Compounds of Formula I in which an R'is lower alkyl can also be prepared by the method described in SectionA, above. ##STR29##

In Formulas IF₁, IG₁ and IG₂, R' is hydrogen or lower alkyl having oneto six carbon atoms. In Formula IG₃, R' is the remaining saturatedportion of the alkene substituent. For illustrative purposes the doublebond is depicted at carbons 2-3; however, other straight and branchedchain lower alkene substituents can be obtained by the method shown inReaction Scheme VII from the appropriate correspondingtriphenylphosphine intermediate of Formula IG₂.

As shown in Reaction Scheme VII, a compound of Formula IF₁ reacts withlithium dialkyl cuprate at low temperature to give the compound ofFormula IG₁. The reaction is normally carried out in an etherealsolvent, preferably anhydrous diethyl ether, at about -25° C. to -78°C., preferably about -40° C. The displacement of benzyl bromide withlithium dialkyl cuprate is known in the art, (See Organic Reactions,Vol. 13, pp. 252 and 401, (John Wiley & Sons) and G. H. Posner,Substitution Reactions using Organocopper Reagents; John Wiley & Sons(1980)) and is further illustrated in Example VII, below.

The monobromo compound of Formula IF₁ can be converted to the Wittigsalt of Formula IG₂ by reacting with triphenylphosphine in toluene. Thereaction is preferably carried out between 60° C. to 70° C., preferably65° C., for a period of about 4 to 6, preferably about 5 hours.

A compound of Formula IG₂ reacts with one equivalent of DBU(1,8-diazabicyclo[5,4,0]-undec-7-ene) at from about -40° C. to -60° C.,preferably -50° C., to form the Wittig ylid. Subsequent quenching withan alkyl aldehyde gives the compound of Formula IG₃. The reaction isnormally carried out in dry tetrahydrofuran; and the product isrecovered by conventional means. The compound of Formula IG₃ representsa mixture of both (E) and (Z) enantiomers.

K. Compounds in which R is Imidazolyl (Formula IH)

Compounds of Formula I in which R is imidazolyl (compounds of FormulaIH) can be prepared by the method shown in Reaction Scheme VIII, below:##STR30##

As shown in Reaction Scheme VIII, the compounds of Formula VI areprepared from the corresponding methyl anthranilate derivative ofFormula V. Methyl anthranilate, as well as variously substituted methyl2-amino benzoates (methyl anthranilates) are commercially available, orcan be prepared by treating the corresponding anthranilic acid withdiazomethane in an inert organic solvent such as tetrahydrofuran orpreferably ether at about 0° C., a method that is standard for theformation of methyl esters. Alternatively, variously substituted methyl2-amino benzoates can be prepared by treating the corresponding isatoicanhydride with methanol in the presence of base such as sodium methoxideor dimethylaminopyridine, preferably dimethylaminopyridine, according tothe literature methods such as that reported by M. C. Venuti, Synthesis,266 (1982), R. P. Straiger and E. B. Miller, J. Org. Chem., 24, 1214(1959).

The corresponding unsubstituted or appropriately substitutedanthranilate of Formula V is treated with about 0.5 to 1 equivalent oftrichloromethyl chloroformate in tetrahydrofuran at room temperature fora period of about 1 to 2, preferably about 1.5 hours; the resultingcarbamyl chloride derivative is then refluxed with about 1 to 2equivalents of 4(N-triphenylmethyl)imidazolylmethanol, or anotherappropriate imidazolyl alcohol, in the presence of 5 to 10 equivalentsof a tertiary amine, preferably triethylamine, for a period of about 0.5to 2 hours. The product, a compound of Formula VI, is isolated byconventional means. The compound of Formula IH is obtained by basehydrolysis of the compound of Formula VI to the corresponding carboxylicacid, followed by cyclization with DCC or EDC in an inert solvent suchas dichloromethane or tetrahydrofuran. The cyclization reaction takesplace at room temperature over a period of about 1 to 3 hours, and thefinal product is then isolated by conventional means.

L. Compounds of Formula I in which an R' is Alkyl at the 5-Position anda Second R' is Nitro or Amino at the 7-Position (Formulas IA and IB)

Compounds of Formula I which bear a lower alkyl R' substituent at the5-position and a nitro or amino R' substituent at the 7-position(compounds of Formulas IA and IB) can be prepared by the procedures setforth in Reaction Schemes I and II, or alternatively, by the procedureillustrated below in Reaction Scheme IX. ##STR31##

As shown in Reaction Scheme IX, the appropriately substituted ethylanthranilate of Formula VII is converted to the carbamoyl chloridederivative by treatment with about 0.5 to 1, preferably about 0.75equivalents of trichloromethyl chloroformate in ethyl acetate at roomtemperature for a period of about 2 to 3, preferably 2, hours. Theresulting carbamoyl chloride derivative is then quenched with about afive fold excess of an appropriate alcohol of the formula HOAR (in whichA and R have the definitions given herein), and a base such as pyridineor triethylamine. The product of Formula VIII is isolated byconventional means. Base hydrolysis of the compound of Formula VIII isthen carried out in about a 1:1 mixture of aqueous sodium hydroxide and1,2-dimethoxyethane to give the carboxylic acid of Formula IX. The acidof Formula IX is then cyclized with DCC or EDC in an inert organicsolvent to give the final product of Formula IA, which is then isolatedby conventional means.

The amino substituted compound of Formula IB can then be obtained formthe corresponding nitro substituted compound of Formula IA by theprocedure outlined in Reaction Scheme II.

Preparation of the ethyl anthranilate starting materials of Formula VIIcan be accomplished by the method illustrated in Reaction Scheme X, asfollows: ##STR32##

As shown in Reaction Scheme X, above, the di-nitro phenol derivative ofFormula 2, which is either commercially available of readily prepared bystandard known methods such as those illustrated in Preparation IV,Paragraph A, herein, is converted to the corresponding chloro-compoundof Formula 3 according to the procedure described by B. Boothroyd and E.R. Clark, J. Chem. Soc., p. 1504, London (1953). Details of thisreaction may also be had by reference to Preparation IV, Paragraph B,below. The compound of Formula 3 is then reacted at room temperaturewith about a 10 fold excess of pentan-2,4-dione and about a 3-4 foldexcess of sodium methoxide in the presence of HMPA as solvent, to givethe (2-alkyl-4,6-dinitrophenyl)-diacetylmethane of Formula 4. Thecompound of Formula 4 is then cyclized in concentrated sulphuric acid atabout 100°-120° C., preferably about 110° C. for a period of about 1 to5, preferably about 3 hours, to give the 4-alkyl-6-nitro anthranil ofFormula 5. Details of this procedure may be had by reference to themethod described by I. R. Gambir and S. S. Joshi in the Indian Chem.Soc. Journal, V. 41, pp. 43-46 (1964), which is specifically illustratedin Preparation IV, Paragraph C, herein. Subsequent ring opening bytreating the anthril of Formula 5 with potassium carbonate and ethanolat reflux temperature gives the ethyl 4-nitro-6-alkyl-2-amino benzoateof Formula VII.

Certain compounds of this invention may be converted to theircorresponding pharmaceutically acceptable acid addition salts by virtueof the presence of a basic amine nitrogen. These compounds may beconverted from the free base form to various acid addition salts bytreating with a stoichiometric excess of the appropriate organic orinorganic acid, such as, for example, phosphoric, pyruvic, hydrochloricor sulfuric acid and the like. Typically, the free base is dissolved ina polar organic solvent such as p-dioxane or dimethoxyethane, and theacid added thereto. The temperature is maintained between about 0° C.and 50° C. The resulting acid addition salt precipitates spontaneouslyor may be brought out of solution with a less polar solvent.

The acid addition salts of the compounds of Formula may be decomposed tothe corresponding free base by treating with a stoichiometric amount ofa suitable base, such as potassium carbonate or sodium hydroxide,typically in the presence of aqueous solvent, and at a temperature ofbetween about 0° C. and 50° C. The free base form is isolated byconventional means, such as extraction with an organic solvent.

Acid addition salts of the compounds of the invention may beinterchanged by taking advantage of differential solubilities of thesalts, volatilities or acidities of the acids, or by treating with anappropriately loaded ion exchange resin. For example, the interchange iseffected by the reaction of a salt of the compounds of Formula I with aslight stoichiometric excess of an acid of a lower pKa than the acidcomponent of the starting salt. This invention is carried out at atemperature between about 0° C. and the boiling point of the solventbeing used as the medium for the procedure.

In summary, then, the compounds of Formula I can be prepared by thefollowing last-step procedures:

I. Cyclization of a compound of Formula II with a phenoxy-, cycloalkoxy-or alkoxy chloroformate to give a compound of Formula IA;

II. Conversion of a nitro-substituted compound of Formula IA to give acompound of Formula IB;

III. Acylation of an amine-substituted compound of Formula IA or IB togive a compound of Formula IC;

IV. Carboalkoxylation of an amino-substituted compound of Formula IA orIB to give a compound of Formula ID;

V. Conversion of an amino-substituted compound of Formula IA or IB togive a compound of Formula IE;

VI. Halogenation of a compound of Formula I to give a compound ofFormula IF₁ or IF₂.

VII. Reaction of a compound of Formula IF₁ with a lithium dialkylcuprate to give a compound of Formula IG₁.

VIII. Reaction of a compound of Formula IF₁ with triphenylphosphine togive a compound of Formula IG₂.

IX. Reaction of a compound of Formula IG₂ with1,8-diazabicyclo[5,4,0']-undec-7-ene and an alkyl aldehyde to give acompound of Formula IG₃.

X. Reaction of a compound of Formula I with a stoichiometric excess ofan acid to give a pharmaceutically acceptable non-toxic acid additionsalt.

XI. Reaction of a salt of a compound of Formula I with a base to givethe corresponding free base.

Utility and Administration

The compounds of Formulas I and A have been shown in standard laboratorytests to inhibit a variety of physiologic enzymes, particularly serineproteases, including human leukocyte elastase, human thrombin, humanurokinase, porcine acrosin, porcine pancreatic elastase, bovinecathepsin B, bovine chymotrypsin, and bovine trypsin. Accordingly, thecompounds of the invention, their salts, esters, and/or pharmaceuticalcompositions thereof, may be used in inhibiting, preventing, orcontrolling physiologic conditions and disease states in animals whichare known to involve enzymes, or may be used as contraceptives.

Knowledge of the roles of enzymes in a wide variety of diseases isconstantly growing. Recent reviews of the state of the art include"Protein Degradation in Health and Disease", Ciba Foundation Symposium75, Excerpta Medica, Amsterdam, 1980; "Proteinases in Mammalian Cellsand Tissues", A. J. Barrett, ed., North Holland Publishing Company,Amsterdam, 1977; and "Proteases and Biological Control", E. Reich, D. B.Rifkin and E. Shaw, eds., Cold Spring Harbor Laboratory, 1975.

Experimental evidence has revealed the roles of many enzymatic pathwaysin various physiologic conditions and disease states. Plasminogenactivator (PA), a serine protease, causes the conversion of plasminogento plasmin which in turn is responsible for fibrinolysis. This processis implicated in a number of systems requiring controlled localproteolysis, including inflammation (J. D. Vassalli, et al. Cell, 8, 271[1976]), and cell migration and tissue remodeling, J. E. Valinski, Cell,25, 471 (1981). The production and secretion of PA is also correlatedwith certain human disorders such as arthritis (Neats, et al., Nature[London], 286, 891, 1980; Hamilton, et al., J. Exp. Med., 155, 1702[1982]) and the expression of transformed phenotypes, D. B. Rifkin, etal., in Proteases and Biological Control, D. Rifkin, E. Reich, E. Shaw,eds., Cold Spring Harbor, 1975, pp. 841-847.

There is considerable evidence that plasminogen activator (such asurokinase), leukocyte elastase, and/or related enzymes play a role intumor cell metastasis (Salo,, et al., Int. J. Cancer, 30, 669-673, 1973;Kao, et al., Biochem. Biophys., Res. Comm., 105, 383-389, 1982; Powers,J. C., in Modification of Proteins, R. E. Feeney and J. R. Whitaker,eds., Adv. Chem. Ser. 198, Amer. Chem. Soc., Washington, D.C., pp.347-367, 1982), suggesting that compounds of this invention may haveanti-metastatic activity.

Other evidence suggests an antiparasitic role for the compounds of thisinvention (Aoki, T., et al., Mol. Biochem., Parasitol, 8, 89-97, 1983).

Pulmonary emphysema is a disease characterized by a progressive loss oflung elasticity due to the destruction of lung elastin and alveoli. Itis widely held that the destructive changes in lung parenchymaassociated with pulmonary emphysema are mediated in large part byunrestrained proteolytic activity in lung connective tissue. (A. Janoff,Chest, 83, 54-58 [1983]. A number of proteases have been shown to induceemphysematous lesions in animals when instilled in lungs (V. Marco, etal., Am. Rev. Respir. Dis., 104, 595-8, 1971; P. D. Kaplan, J. Lab.Clin, Med., 82, 349-56 (1973)). In particular, human leukocyte elastasehas been shown to produce emphysema in animals (A. Janoff, ibid, 115,461-78 (1977)). Prophylactic administration of an inhibitor of elastasesignificantly diminishes the extent of elastase induced emphysema inhamsters (J. Kleinerman, et al., ibid, Am. Rev. Respir. Dis., 121,381-7, 1980).

Leukocyte elastase and other mediators of inflammation appear to play arole in such acute and high-risk diseases as mucocutaneous lymph nodesyndrome (Rieger, et al., Eur. J. Pediatr., 140, 92-97, 1983), and adultrespiratory distress syndrom (Stockley, R. A., Clinical Science, 64,119-126, 1983; Lee, et al., N. Eng. J. Med., 304, 192-196, 1981;Rinaldo, ibid, 301, 900-909, 1982.

Oral anticoagulants are some of the most important drugs for theprevention and treatment of a variety of venous and, to a lesser extent,arterial thromboembolic disorders (R. A. O'Reilly in "ThePharmacological Basis of Therapeutics", 6th Ed., A. G. Goodman, L. S.Goodman, A. Gilman, eds., 1980). The enzymes that participate in thecascade leading to blood coagulation are proteases. The coagulation ofblood entails the formation of fibrin by the interaction of more than adozen proteins in a cacading series of proteolytic reactions. Inhibitionof these proteinases should block fibrin formation and hence inhibitcoagulation. For example, inhibition of thrombin limits the formation offibrin and is regarded as an approach to thromboembolic therapy.

However, anticoagulants that are in current use and that affect clottingfactors do not have a direct onset of action. Consequently, prothrombintime must be monitored, as the degree of Vitamin K antagonism Variesfrom individual to individual.

Thus there is a critical need for new anticoagulants which have a directonset of action. Pulmonary embolism (PE), for example, is a commoncomplication that usually affects patients who are hospitalized forother medical or surgical problems (A. A. Sasahara, et al., JAMA, 249,2945 (1983) and references therein). The mortality of undiagnosed andtherefore untreated PE is relatively high, ranging from about 18% to35%. Patients undergoing total hip or knee replacement are at extremelyhigh risk for development of deep vein thrombosis, with a reportedincidence of 45% to 70% in untreated patients S. Sagar, et al., Lancet,1, 1151 (1978)).

Pancreatitis is a disease which affects large numbers of people includngpatients having acute alcoholic, acute biliary traumatic andpost-operative pancreatitis. Furthermore, with the high incidence ofalcoholism, 10,000,000 alcoholics in the U.S. alone, acute and chronicrelapsing pancreatitis are seen with increasing frequency. Geokas, etal. has proposed that an effective therapy for acute pancreatitis mightbe achieved by the use of "a combination of a low molecular weightspecific active-site inhibitors for trypsin, chymotrypsin, andelastase", (Am. J. Pathol, 1981, 105, 31-39).

Enzymes possessing cathepsin B-like activity have also receivedattention because of their extracellular release by neoplasticepithelial cells (Pietras, J. Histochem. Cytochem., 29, 440-450 1981),their presence in the interstitial fluid (Sylven, et al., Cancer Res.,20, 831-836, 1960; Eur. J. Cancer., 463-474, 1968; Virchows Arch. B.Cell Pathol., 17, 97-112, 1974), and in the invasive zone of malignanttissue (R. R. Labrosse, Mol. Cell Biochem., 19, 181-189, 1978) and theirenhanced secretion by malignant and metastatic tumors (B. F. Sloane,Cancer Res., 42, 980-986, 1982).

Proteolytic cleavage of precursors is an essential step in thereplication of many animal viruses, and there is considerable evidencethat protease inhibitors can be effective anti-viral agents (Korant, B.D., (1975) in "Proteases and Biological Control"). Such viruses includeinfluenza (Chirov, O. P. et al. (1981) Vopr. Virusol. 6, 677-687). InSendai virus, for example, a host trypsin-like protease is essential forinfectivity (Scheid, A., and Choppin, P. (1975) in "Proteases andBiological Control"). It is reasonable then that compounds of thisinvention could play a role in amelioration of viral diseases.

Acrosin is a unique serine proteinase which is present in mammaliansperm acrosomes (L. J. D. Zaneveld (1975) in "Proteases and BiologicalControl", pp. 683-706; R. F. Parrish, Int. J. Biochem., 10, 391-395(1979)). Since acrosin activity is required for fertilization, it is arational target for birth control. Further, the inhibition of acrosin isknown to prevent fertilization (Zaneveld, L. J. D., et al., (1979),Biol. Repr. 20, 1045-1054), supporting a role for acrosin inhibitors ascontraceptives.

Initial screening tests to determine enzyme-inhibitory potential can beperformed with commercially available enzyme substrates such as peptidylamides of 4-methyl-7-amino coumarin or 4-nitroaniline. The assays areperformed by mixing the substrate and enzyme of interest in anappropriate buffer, and monitoring the rate of enzyme inhibitionspectrophotometrically. The reaction rate is monitored continuouslyeither by fluorescence (for coumarin substrates) or absorbance (fornitroanilide substrates) until a constant reaction rate is established.A solution of the compound to be tested in an appropriate solvent, suchas a 5 to 20 millimolar solution in dimethyl sulfoxide, is then added,and the increase in fluorescence or absorbance is monitored until a newstable rate is achieved. This is repeated for several concentrations oftest compound solution, and the inhibition constant is calculated bynon-linear multiple regression fit to the appropriate equation. Thecompounds of Formula I have been tested in assays of this type and havedemonstrated marked inhibitory activity against human leukocyteelastase, human thrombin, human urokinase, porcine acrosin, porcinepancreatic elastase, bovine chymotrypsin and bovine and human trypsin.Some of the compounds of Formula I have also been tested and shown to beactive in inhibiting the degradation of basement membrane bymacrophages, tumor cells and elastase. More detailed descriptions ofseveral of these assays may be found in the Examples, below.

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for systemically activetherapeutic medicaments. These methods include oral, parenteral andotherwise systemic, aerosol or topical forms.

Depending on the intended mode of administration, the compositions usedmay be in the form of solid, semi-solid or liquid dosage forms, such as,for example, tablets, suppositories, pills, capsules, powders, liquids,aerosols, suspensions, or the like, preferably in unit dosage formssuitable for single administration of precise dosages. The compositionswill include a conventional pharmaceutical carrier or excipient and anactive compound of Formula I or A or the pharmaceutically acceptablesalts thereof and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

For solid compositions, conventional non-toxic solid carriers includes,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like may be used. The activecompound as defined above may be formulated as suppositories using, forexample, polyalkylene glycols, for example, propylene glycol, as thecarrier. Liquid pharmaceutically administerable compositions can, forexample, be prepared by dissolving, dispersing, etc. an active compoundas defined above and optional pharmaceutical adjuvants in a carrier,such as, for example, water, saline, aqueous dextrose, glycerol,ethanol, and the like, to thereby form a solution or suspension. Ifdesired, the pharmaceutical composition to be administered may alsocontain minor amounts of nontoxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents and the like, for example,sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate,triethanolamine oleate, etc. Actual methods of preparing such dosageforms are known, or will be apparent, to those skilled in this art; forexample, see Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa, 15th Edition, 1975. The composition or formulationto be administered will, in any event, contain a quantity of the activecompound(s) in an amount effective to alleviate the symptoms of thesubject being treated.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, etc.

For the compounds of Formula I, either oral or nasal (bronchial)administration is preferred, depending on the nature of the disorderbeing treated.

For oral administration, a pharmaceutically acceptable non-toxiccomposition is formed by the incorporation of any of the normallyemployed excipients, such as, for example pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium, carbonate, and the like. Suchcompositions take the form of solutions, suspensions, tablets, pills,capsules, powders, sustained release formulations and the like. Suchcompositions may contain 1%-95% active ingredient, preferably 25-70%.

Oral and nasal administration to the lungs can also be effected byaerosol delivery forms. For aerosol administration, the activeingredient is preferably supplied in finely divided form along with asurfactant and a propellant. Typical percentages of active ingredientsare 0.01 to 20% by weight, preferably 0.04 to 1.0%.

Surfactants must, of course, be non-toxic, and preferably soluble in thepropellant. Representative of such agents are the esters or partialesters of fatty acids containing from 6 to 22 carbon atoms, such ascaproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,olestearic and oleic acids with an aliphatic polyhydric alcohol or itscyclic anhydride such as, for example, ethylene glycol, glycerol,erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derivedfrom sorbitol (the sorbitan esters sold under the trademark "Spans") andthe polyoxyethylene and polyoxypropylene derivatives of these esters.Mixed esters, such as mixed or natural glycerides may be employed. Thepreferred surface-active agents are the oleates or sorbitan, e.g., thosesold under the trademarks "Arlacel C" (Sorbitan sesquioleate), "Span 80"(sorbitan monooleate) and "Span 85" (sorbitan trioleate). The surfactantmay constitute 0.1-20% by weight of the composition, preferably 0.25-5%.

The balance of the composition is ordinarily propellant. Liquefiedpropellants are typically gases at ambient conditions, and are condensedunder pressure. Among suitable liquefied propellants are the loweralkanes containing up to five carbons, such as butane and propane; andpreferably fluorinated or fluorochlorinated alkanes, such as are soldunder the trademark "Freon." Mixtures of the above may also be employed.

In producing the aerosol, a container equipped with a suitable valve isfilled with the appropriate propellant, containing the finely dividedactive ingredient and surfactant. The ingredients are thus maintained atan elevated pressure until released by action of the valve.

For topical administration, these compositions comprise an effectiveamount of a compound of this class in admixture with a pharmaceuticallyacceptable non-toxic carrier. A suitable range of composition would be0.1%-10% active ingredient, and the balance carrier, preferably 1-2%active ingredient. The concentration of active ingredient inpharmaceutical compositions suitable for topical application will varydepending upon the particular activity of the compound used inconjunction with the condition and subject to be treated. Suitablecarriers or medicament vehicles for topical application of thesecompounds include creams, ointments, lotions, emulsions, solutions andthe like.

For example, a suitable ointment for topical application of compounds ofthe invention contains 15 to 45 percent of a saturated fatty alcoholhaving 16 to 24 carbon atoms such as cetyl alcohol, stearyl alcohol,behenyl alcohol, and the like and 45 and 85 wt. percent of a glycolsolvent such as propylene glycol, polyethylene glycol, dipropyleneglycol, and mixtures thereof. The ointment can also contain 0 to 15 wt.percent of a plasticizer such as polyethylene glycol, 1,2,6-hexanetriol,sorbitol, glycerol, and the like; 0 to 15 wt. percent of a couplingagent such as a saturated fatty acid having from 16 to 24 carbon atoms,e.g., stearic acid, palmitic acid, behenic acid, a fatty acid amidee.g., oleamide, palmitamide, stearamide, behenamide and an ester of afatty acid having from 16 to 24 carbon atoms such as sorbitolmonostearate, polyethylene glycol monostearate, polypropylene glycol orthe corresponding mono-ester of other fatty acids such as oleic acid andpalmitic acid; and 0 to 20 wt. percent of a penetrant such as dimethylsulfoxide or dimethylacetamide.

The amount of active compound administered will of course, be dependenton the subject being treated, the severity of the affliction, the mannerof administration and the judgement of the prescribing physician.However, an effective dosage is in the range of 1-100 mg/kg/day,preferably about 25 mg/kg/day. For an average 70 kg human, this wouldamount to 70 mg-7 g per day, or preferably about 1.5 g/day.

The following examples serve to illustrate the invention. They shouldnot be construed as narrowing or limiting its scope.

PREPARATION I Preparation of Substituted anthranilic acids of Formula IIA. Preparation of 4-ethyl anthranilic acid and 6-ethyl-anthranilic acid

4-ethyl anthranilic acid and 6-ethyl anthranilic acid were preparedaccording to Baker's procedure, as described in J. Org. Chem. 17, 141,(1952) and further detailed below.

(i) Preparation of m-ethyl-alpha-isonitrosoacetanilide.

In a 5 liter round-bottom flask equipped with overhead stirrer andcondensers were placed 74.2 gm. of chloral dihydrate and 900 ml ofwater. To this solution was then added, sequentially, 107.2 gm ofanhydrous sodium sulfate, a solution of 50 gm of m-ethyl anilinedissolved in 248 ml of water and 42 ml of concentrated hydrochloricacid, and lastly, a solution of 90.8 gm of hydroxylamine hydrochloridein 412 ml of water. The mixture was slowly heated over a period of 45minutes to a temperature of 95° C. The heating mantle was then removedand the flask rapidly cooled to room temperature by immersion in anice-bath. The crude isonitrosoacetanilide was collected by suctionfiltration and washed with water. The product was then further purifiedby the following procedure: the crude isonitrosoacetanilide wasdissolved in 500 ml of a 4M sodium hydroxide solution, transferred to aseparatory funnel and washed with ether (3×300 ml). The alkaline phasewas then treated with charcoal, filtered through Celite and stronglyacidified with concentrated hydrochloric acid. The precipitatedm-ethyl-alpha-isonitrosoacetanilide was collected by filtration anddried under vacuum, mp. 140°-142° C.

(ii) Preparation of 4-ethyl and 6-ethyl isatin.

A 1 liter round-bottom flask containing 370 ml of concentrated sulfuricacid and 30 ml of water was heated to 60° C.

m-Ethyl-alpha-isonitrosoacetanilide (64 gm) was added at such a rate asto maintain the temperature between 60° and 65° C. After the additionwas completed, the mixture was heated to 80° C. for 10 minutes. Theflask was then cooled to room temperature and poured onto 8 to 10 timesits volume of ice. After standing for one-half hour, the crude isatinmixture was collected by filtration and washed well with water. Thecrude extract was then dissolved in about 300 ml of a 3M sodiumhydroxide solution by heating on a steam bath, treated with charcoal andfiltered through Celite. On acidification to pH 6-7 with concentratedhydrochloric acid, a gummy material appeared and was removed byfiltration through Celite. The solution was then acidified to pH 4 andthe 4-ethyl isatin was collected by filtration and washed with water:Yield 14.6 gm, mp 128°-136° C. The cooled filtrate was then stronglyacidified with concentrated hydrochloric acid and collected byfiltration to give the 6-ethyl isatin: Yield 16.4 gm (28%), mp.171°-173° C.

(iii) Preparation of 4-ethyl-anthranilic acid

In a 500 ml flask, was placed 16.84 gm of 6-ethyl isatin which wascovered with 216 ml of 1.5M sodium hydroxide solution. With stirring,the mixture was warmed to 50° C. Heating was discontinued and thesolution was treated with a 30% solution of hydrogen peroxide (24 ml)which was added at such a rate to maintain the temperature at between50° to 65° C. The mixture was left to slowly cool to room temperatureand was then acidified to pH 4 with concentrated hydrochloric acid. Theprecipitated product was then collected by filtration: mp. 117°-120° C.;yield 8.93 gm.

(iv) Preparation of 6-ethyl-anthranilic acid.

Oxidation of 9.6 gm of 4-ethyl isatin according to the method describedin (iii), above, gave 7.3 gm of the title compound: mp. 99°-104° C.

B. In a similar manner, but replacing m-ethyl aniline with otheranilines, the following exemplary compounds of Formula VII are prepared.

6-butyl-anthranilic acid;

4-iodo-anthranilic acid;

6-methyl anthranilic acid;

4,6-dimethyl-anthranilic acid;

3,5-dimethyl-anthranilic acid;

3,5.6-trimethylanthranilic acid;

3,6-dimethylanthranilic acid;

5-butyl-anthranilic acid;

4-methoxy-6-methyl-anthranilic acid; and

4,6-dichloro-anthranilic acid.

C. Preparation of 6-methoxyanthranilic acid by reduction of thecorresponding aromatic nitro compounds was carried out in accordancewith Paquette's procedure, J. Am. Chem. Soc., 99, 3734, (1981), mp.71°-75° C., which can also be used to prepare other 6-alkoxy anthranilicacids.

D. Preparation of 4-nitro-6-iodo-anthranilic acid and4-nitro-6-bromo-anthranilic acid are prepared according to the procedureof I. R. Gambir and S. S. Joshi, Indian Chem. Soc., Journal 43-46,(1964). In a similar manner, the following compounds can be prepared:

4-nitro-6-ethyl-anthranilic acid;

4-nitro-6-methyl-anthranilic acid; and

4-nitro-6-propyl-anthranilic acid.

E. Preparation of 5-Alkoxyanthranilic Acid and 5-Alkylthioanthranilicacid

5-Methoxyanthranilic acid and 4-methylthioanthranilic acid were preparedaccording to the procedure of J. W. Tilley, J. Kudless, R. W. Kierstead,Organic Preparations Procedure Int. 13(3-4), 189-196 (1981).

In a similar manner the following cmpounds are prepared:

5-ethoxy-anthranilic acid;

5-butoxy-anthranilic acid;

5-hexoxy-anthranilic acid;

5-isopropoxy-anthranilic acid,

5-ethylthio-anthranilic acid.

5-methylthio-anthranilic acid;

5-n-pentylthio-anthranilic acid;

5-n-propylthio-anthranilic acid.

F. Preparation of 4-N,N-Dimethylamino-Anthranilic Acid

4-N,N-dimethylamino-anthranilic acid was prepared according to theprocedure of D. H. Klaubert, J. H. Sellstedt, C. J. Guinosso, R. J.Capetola, J. S. C. Bell, J. Med. Chem., 1981, 24, 742-748.

In a similar manner the following compounds are prepared:

4-N,N-diethylamino-anthranilic acid; and

4-N,N-dipropylamino-anthranilic acid.

PREPARATION II Phenyl-, Cycloalkyl- and Alkyl-Chloroformates of FormulaIII A. Preparation of N-Butyl Chloroformate

Phosgene was passed into dry ether until saturated (15-20% w/v). n-Butylalcohol (10 gm) in dry ether (50 ml) was treated with ethereal phosgene(1.1 mol.) at room temperature until the reaction was complete. Removalof the solvent in vacuo gave n-butyl chloroformate in quantitativeyield. In representative cases these compounds were purified bydistillation, but this is not necessary for the subsequent cyclization.For the preparation of the chloroformates of hindered alcohols,quinoline can be added as a catalyst, quinoline hydrochloride beingsubsequently removed by filtration.

B. Thus, in a similar manner, but replacing the n-butyl alcohol withn-hexyl alcohol, s-butyl alcohol, isobutyl alcohol, n-pentyl alcohol,benzyl alcohol, phenol, phenethyl alcohol, 1-phenyl-2-methyl-propanol,1-phenyl-3-methyl-2-butanol, 1-phenyl-pentanol, salicyl alcohol,procatechol, citronehol, (-)-menthol, cholestanol, and the like, thefollowing compounds of Formula III are prepared:

methyl chloroformate;

ethyl chloroformate;

n-propyl chloroformate;

isopropyl chloroformate;

n-butyl chloroformate;

s-butyl chloroforomate;

isobutyl chlorormate;

n-pentyl chlorformate;

n-hexyl chloroformate;

n-octyl chloroformate;

phenyl chloroformate;

benzyl chloroformate;

phenethyl chloroformate;

1-phenylpentyl chloroformate;

1-phenyloctyl chloroformate;

1-phenyl-2-methyl-propyl chloroformate;

1-phenyl-3-methyl-2-butyl chloroformate; 1;

1-(3-methoxyphenyl)-octyl chloroformate;

1-(4-isobutoxyphenyl)-heptyl chloroformate;

2-carbomethoxyphenyl chloroformate;

(-)-menthyl chloroformate;

cholesteryl chloroformate;

cyclopropyl chloroformate;

cyclobutyl chloroformate;

cyclohexyl chloroformate;

1-cyclopropyl-ethyl chloroformate;

1-cyclobutyl-methyl chloroformate;

1-cyclohexyl-propyl chloroformate;

1-(2-ethylcyclohexyl)methyl chloroformate;

1-(4-nitrocyclohexyl)-pentyl chloroformate; and

1-(2-dimethylaminobenzyl)chloroformate.

PREPARATION III Acid Anhydrides of Formula IV A. Preparation of butyricanhydride

Molar quantities of butyric acid and butyryl chloride are heatedtogether on a water-bath for 1 hour and then boiled for 7 hours in anoil-bath. Butyric anhydride, b.p. 198°-199° C./765 mm, is obtained ondistillation of the resulting mixture.

B. In a similar manner, but starting instead with acetyl chloride andsodium acetate, acetic anhydride is obtained.

C. In like manner, other symmetrical and mixed acid anhydrides areobtained from the corresponding free acids and acid chlorides, oralternatively from the acid chlorides and alkali salts of the carboxylicacids, including;

hexanoic anhydride; and

acetic propionic anhydride.

PREPARATION IV Ethyl 2-amino-6-ethyl-4-nitrobenzoate and RelatedCompounds of Formula VII A. Preparation of 2,4-dinitro-6-ethylphenol

(i) Concentrated sulfuric acid (25 g) was added to 2-ethylphenol (25 g,Aldrich) with swirling. The solution was heated on a steam bath for onehour, cooled and 25 ml water added. The solution was placed in adropping funnel and added dropwise to 70% nitric acid (40 g), withcooling provided by an ice-salt bath cooled by glycol. The solution inthe dropping funnel was added over 1.5 to 2 hours, with the temperaturekept below 0° C. The resulting mixture was stirred at 0° C. for threehours, the ice bath removed, and the mixture further stirred overnightat room temperature. The mixture was then heated on a steam bath for onehour, cooled and 50 ml of water added. Following extracted with diethylether, the ether layer was washed with brine, dried over MgSO₄ andevaporated to a dark oil which was columned on silica gel using 10%ethyl acetate-petroleum ether. The combined filtrates gave ayellow-orange oil, which solidified upon being pumped dry to give2,4-dinitro-6-ethylphenol, yield 34 gm.

(ii) Proceeding in a similar matter but starting instead with2-propylphenol, 2-4-dinitro-6-propylphenol was obtained as an oil.

(iii) Similarly prepared are the following compounds of Formula 2:

2,4-dinitro-6-isopropylphenol;

2,4-dinitro-6-butylphenol; and

2,4-dinitro-6-isobutylphenol.

B. Preparation of 1-chloro-2,4-dinitro-6-ethylbenzene

(i) 2,4-Dinitro-6-ethylphenol (10 g) was placed in a 250 ml round bottomflask, and phosphorous oxychloride (60 ml, Fisher) was added.N,N-diethylamiline (15 ml, Aldrich was added portionwise, and themixture became hot. The flask was placed under a condenser equiped witha drying tube heated on a steam bath for two hours and cooled. Themixture was then carefully poured onto ice, with stirring and extractedwith ethyl acetate. The ethyl acetate layer was washed with brine, driedover MgSO₄ and evaporated in a dark oil which was columned on silica gelusing 10% ethyl acetate petroleum ether. The evaporated filtrates gave areddish oil, which solidified upon being pumped dry. The solid wasrecrystallized from ethyl acetate-petroleum ether to give pale yellowneedles of 1-chloro-2,4-dinitro-6-ethylbenzane.

Yield: 8.1 g, m.p. 41°-44° C.

IR: 3400 cm, 3090 cm, 2980 cm, 1800 cm (w), 1540 cm, 1345 cm

(ii) Proceeding in a similar manner, the following compounds of Formula3 were prepared:

1-chloro-2,4-dinitro-6-methylbenzene mp;

1-chloro-2,4-dinitro-6-propylbenzene oil.

(iii) Similarly, but starting instead with other appropriatecorresponding compounds of Formula 2, the following compounds of Formula3 are prepared:

1-chloro-2,4-dinitro-6-isopropylbenzene;

1-chloro-2,4-dinitro-6-butylbenzene; and

1-chloro-2,4-dinitro-6-isobutylbenzene.

C. Preparation of (6-ethyl-2,4-dinitrophenyl)-diacetylmethane

(i) Sodium methoxide (8.16 g) was placed in a flask containhexamethylphosphoramide (50 ml, Aldrich). 2,4-pentanedione (50 ml,Aldrich) was added and the mixture stirred while gently heated with aheating mantle (Variac 25/140) for thirty minutes.

6-Ethyl-2,4-dinitrochlorobenzene (10 g) in some dry tetrahydrofuran wasadded, and the mixture heated at the same setting for two more hours.The reaction mixture was cooled and partitioned between ethyl acetateand 5N hydroclhoric acid.

The ethyl acetate layer was washed with 5N HCl, water and brine, driedover MgSO₄ and evaporated to a dark oil. 350 ml of 10% ethylacetate-petroleum ether was added, and a pale ywellow solid precipitatedout. This was colummed on silica gel using 20% ethyl acetate-petroleumether to give 5.4 g of (2,4-dinitro-6-ethyl)-diacetylmethane,

m.p.: 126°-128° C.,

IR: 3100 cm, 2980 cm, 1525 cm, 1345 cm.

(ii) Proceeding in the same manner, the following compounds of Formula 4were prepared:

(6-methyl-2,4-dinitrophenyl)-diacetulmethane, m.p. 145°-147° C.; and

(6-propyl-2,4-dinitrophenyl)-diacetylmethane, m.p. 147°-147.5° C.

(iii) In like manner, but substituting other corresponding compounds ofFormula 3, the following compounds of Formula 4 are prepared:

(6-isopropyl-2,4-dinitrophenyl)-diacetylmenthane;

(6-butyl-2,4-dinitrophenyl)-diacetylmenthane; and

(6-isobutyl-2,4-dinitrophenyl)-diacetylmethane.

B. Preparation of 4-ethyl-6-nitro-anthranil

(i) (6-ethyl-2,4-dinitrophenyl)-diacetylmethane (5 g) was dissolved inconcentrated sulfuric acid and heated to 90°-110° C. in an oil bath forthree hours. The mixture was poured onto ice, with stirring, andextracted with methylene chloride. The resulting emulsion was filteredthrough Celite to separate the layers. The methylene chloride layer wasdried over MgSO₄ and evaporated to a dark solid which was re-dissolvedin some methylene chloride and colummed on silica using 10% ethylacetate-petroleum ether. Evaporation of the filtrate 2.57 g of4-ethyl-6-nitro-anthranil as an orange solid,

m.p 69°-72° C.,

IR: 3140 cm, 3100 cm, 2970 cm, 1550 cm, 740 cm.

(ii) Proceeding in the same manner, but starting with other appropriatecompounds of Formula 4, the following compounds of Formula 5 wereprepared:

4-methyl-6-nitro-anthranil, m.p. 158°-160° C.; and

4-propyl-6-nitro-anthranil, m.p. 82°-84° C.

(iii) In like manner, the following compounds of Formula 5 are prepared:

4-isopropyl-6-nitro anthranil;

4-butyl-6-nitro anthranil; and

4-isobutyl-6-nitro anthranil

E. Preparation of Ethyl-2-amino-6-ethyl-4-nitrobenzoate

(i) 4-ethyl-6-nitro anthranil (2 g) was refluxed for three hours inethanol with potassium carbonate. The reaction mixture was cooled,filtered and evaporated to a dark oily solid which was dissolved inethyl acetate and dried. The residue was re-dissolved in methylenechloride and columned on silica gel using 15% ethyl acetate-petroleumether. The residue from the filtrate was recrystallized from methylenechloride-petroleum ether to give 1.9 gethyl-2-amino-6-ethyl-4-nitrobenzoate, m.p. 68°-70° C., IR: 3490 cm,3380 cm, 3080 cm, 2980 cm, 1690 cm, 1620 cm, 1515 cm, 1350 cm.

(ii) Proceeding in the same manner, but starting instead with otherappropriate, 4-alkyl-6-nitro anthranils of Formula 5, the followingcompounds of Formula VII were prepared:

ethyl 2-amino-6-methyl-4-nitrobenzoate, m.p. 67°-68° C.; and

ethyl 2-amino-6-propyl-4-nitrobenzoate, m.p. 78°-79° C.

(iii) In like manner, the following compounds of Formula VII areprepared:

ethyl-2-amino-6-isopropyl-4-nitrobenzoate;

ethyl-2-amino-6-butyl-4-nitrobenzoate; and

ethyl-2-amino-6-isobutyl-4-nitrobenzoate.

PREPARATION V A. Preparation of Ethyl2-carbethoxyamino-6-ethyl-4-nitro-benzoate and Related Compounds ofFormula VIII

A solution of ethyl-2-amino-6-ethyl-4-nitro-benzoate (400 mg) in ethylacetate (10 ml) was added dropwise to a solution of trichloromethylchloroformate in ethylacetate (10 ml). A precipitate was formedimmediately which dissolved on prolonged sitrring for 21/2 hours. Asolution of ethanol (5 ml) and triethylamine (5 ml) was added. Ayellowish precipitate was formed. After 1 hour the solution waspartitioned between ethylacetate and water. The ethylacetate layer waswashed with brine solution, dried over magnesium sulfate and evaporatedto a reddish oil. The material was further purified by columnchromatography on silica gel (10% EtOAc:pet.ether) to give a solidmaterial,

m.p. 47°-48° C.,

IR: 1530, 1710, 1740, 1610 cm.

B. Proceeding in a similar manner but replacing the2-amino-6-ethyl-4-nitro-benzoate with other appropriate correspondingcompounds of Formula VII, prepared as described in Preparation IV andreplacing the ethanol with other appropriate alcohols, as desired, thefollwoing compounds of Formula VIII are prepared:

ethyl 2-carbobenzyloxyamino-6-ethyl-4-nitro-benzoate;

ethyl 2-carboethoxyamino-6-methyl-4-nitro-benzoate;

ethyl 2-carboisopropoxyamino-6-propyl-4-nitro-benzoate; and

ethyl 2-carbocyclopropyloxyamino-6-isobutyl-4-nitro-benzoate.

PREPARATION VI A. Preparation of 2-carboethoxyamino-4-nitro-6-ethylbenzoic acid and Related Compounds of Formula IX

A solution of ethyl 2-carboethoxyamino-4-nitro-6-ethyl-benzoate intetrahydrofuran (10 ml) and sodium hydroxide (20 ml, 10%) was stirred atroom temperature for 20 hours. The solution was extracted with ethylacetate. The aqueous layer was acidified to pH=1 with 6M HCl, and thenimmediately extracted with ethylacetate. The ethyl acetate extract waswashed with water and dried over magnesium sulphate. Solvent evaporationgave a solid which was further recrystallized from methylenechloride:petroleum ether to yield 2-carboethoxyamino-4-nitro-6-ethylbenzoic acid,

m.p. 121°-123° C. as orange crystals;

IR: 1665, 1720, 1620, 1510, 2500-3200(br), 3500 cm.

B. Proceeding in a similar manner, but replacing the2-carboethoxyamino-4-nitro-6-ethyl-benzoate with other compounds ofFormula VIII, the preparation of which is described in Preparation Vabove, the following compounds of Formula IX are prepared:

2-carbobenzyloxyamino-6-ethyl-4-nitro-benzoic acid;

2-carboethoxyamino-6-methyl-4-nitro-benzoic acid;

2-carboisopropoxy-amino-6-propyl-4-nitro-benzoic acid;

2-carboisopropoxyamino-6-butyl-4-nitrobenzoic acid; and

2-carbocyclopropyloxyamino-6-isobutyl-4-nitrobenzoate.

EXAMPLE I A. Synthesis of 2-ethoxy-4H-3,1-benzoxazin-4-one, and RelatedCompounds of Formula IA

To a solution of anthranilic acid (0.1 mol., 13.71 gm) in dry pyridine(100 ml) at room temperature under anhydrous conditions was added ethylchloroformate (4 equiv., 38.25 ml) in a dropwise manner over 15 minutes.After stirring for 2 hours, excess pyridine was removed under reducedpressure at 40° C. (bath temperature) and the residue was stirredvigorously in ice cold water (250 ml) for 15 minutes. The pale yellowpowder was collected by filtration, washed with water (100 ml), andair-dried to give 18.6 gm of crude product. The crude product wastreated with active charcoal (2 gm) in ethyl acetate (150 ml) to afforda white solid after removal of solvent. Recrystallization fromEtOAc-ether gave 17.02 (89%) of the title compound,2-ethoxy-4H-3,1-benzoxazin-4-one, as colorless crystals; m.p. 88°-90.5°C.; IR (KBr): ν_(max) 1760 cm⁻¹ (C═O), 1630 cm⁻¹ (C═N); H'NMR (CDCl₃);δ1.46 ppm (t, J=7.1 Hz, 3H,)CH₂ CH₃), 4.53 ppm (q, J=7.1 Hz, 2H, OCH₂CH₃), 7.30-8.20 ppm (m, 4H, aromatic protons).

B. In a similar manner, but substituting other appropriately substitutedanthranilic acids (which may be commerially obtained or prepared asdescribed in Preparation I, above), for the anthranilic acid, thefollowing substituted compounds of Formula IA were prepared:

2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one, m.p. 104°-105° C.;

2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one, m.p. 89°-91° C.;

2-ethoxy-6-methylthio-4H-3,1-benzoxzazin-4-one, m.p. 67°-68.5° C.;

6-dimethylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 93°-95° C.;

7-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 191°-193° C.;

6,7-dimethoxy-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 169°-170° C.;

2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one, m.p. 106°-109° C.;

7-dimethylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 196°-198° C.;

2-benzyloxy-4H-3,1-benzoxazin-4-one; m.p. 88°-89.5° C.; and

2-benzyloxy-6,7-dimethoxy-4H-3,1-benzoxazin-4-one, m.p. 152°-154° C.

C. In like manner, but replacing the ethyl chloroformate in Paragraph A,above, with other corresponding chloroformates of Formula III, (whichmay be commercially obtained, or prepared as described in PreparationII, above), the following compounds of Formula IA are prepared:

2-methoxy-4H-3,1-benzoxazin-4-one;

2-n-propoxy-4H-3,1-benzoxazin-4-one;

2-isopropoxy-4H-3,1-benzoxazin-4-one;

2-n-butoxy-4H-3,1-benzoxazin-4-one;

2-s-butoxy-4H-3,1-benzoxazin-4-one;

2-isobutoxy-4H-3,1-benzoxazin-4-one;

2-n-pentoxy-4H-3,1-benzoxazin-4-one;

2-n-hexoxy-4H-3,1-benzoxazin-4-one;

2-n-octyloxy-4H-3,1-benzoxazin-4-one;

2-phenyloxy-4H-3,1-benzoxazin-4-one;

2-benzyloxy-4H-3,1-benzoxazin-4-one;

2-phenethyloxy-4H-3,1-benzoxazin-4-one;

2-phenylbutyloxy-4H-3,1-benzoxazin-4-one;

2-phenyloctyloxy-4H-3,1-benzoxazin-4-one;

2-(1-phenyl-2-methyl-propyl)oxy-4H-3,1-benzoxazin-4-one;

2-(1-phenyl-3-methyl-2-butyl)oxy-4H-3,1-benzoxazin-4-one;

2-(1-(3-methoxyphenyl)octyloxy-4H-3,1-benzoxazin-4-one;

2-(1-(4-isobutoxyphenyl)heptyloxy-4H-3,1-benzoxazin-4-one

2-(4-ethylbenzyl)oxy-4H-3,1-benzoxazin-4-one;

2-(3,5-dimethoxybenzyl)oxy-4H-3,1-benzoxazin-4-one;

2-carbomethoxyphenyloxy-4H-3,1-benzoxazin-4-one;

2-citronyloxy-4H-3,1-benzoxazin-4-one;

2-(-)-methyloxy-4H-3,-benzoxazin-4-one;

2-cholesteryloxy-4H-3,1-benzoxazin-4-one;

2-cyclopropyloxy-4H-3,1-benzoxazin-4-one;

2-cyclobutyloxy-4H-3,1-benzoxazin-4-one;

2-cyclohexyloxy-4H-3,1-benzoxazin-4-one;

2-cyclopropylbutyloxy-4H-3,1-benzoxazin-4-one;

2-(4-methylthiocyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one;

2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one;

2-5-methylhexyloxy)-4H-3,1-benzoxazin-4-one;

2-(3-methyl-4-ethylpentyl)oxy-4H-3,1-benzoxazin-4-one;

2-(2-methylpentyl)oxy-4H-3,1-benzoxazin-4-one;

2-(1-[2-ethylcyclohexyl]methyl)oxy-4H-3,1-benzoxazin-4-one

2-(1-[4-nitrocyclohexyl]pentyl)oxy-4H-3,1-benzoxazin-4-one

2-(1-[2-methylthiocyclopentyl]ethyl)oxy-4H-3,1-benzoxazin-4-one; and

2-(1-dimethylaminobenzyl)oxy-4H-3,1-benzoxazin-4-one;

D. Similarly, but further replacing the unsubstituted anthranilic acidwith appropriate substituted anthranilic acids of Formula II, (which maybe commercially obtained, or can be prepared as described in PreparationI, above), the following compounds of Formula IA are prepared:

2-methoxy-5-methyl-4H-3,1-benzoxazin-4-one;

2-isopropoxy-5-ethyl-4H-3,1-benzoxazin-4-one;

2-n-butoxy-5-methyl-7-methoxy-4H-3,1-benzoxazin-4-one;

2-s-butoxy-5-methylthio-4H-3,1-benzoxazin-4-one;

2-isobutoxy-5-chloro-4H-3,1-benzoxazin-4-one;

2-n-hexoxy-7-ethoxy-4H-3,1-benzoxazin-4-one;

2-n-octyloxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-phenyloxy-5-ethyl-4H-3,1-benzoxazin-4-one;

2-benzyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

2-phenethyloxy-5,7-dimethoxy-4H-3,1-benzoxazin-4-one;

2-phenylbutyloxy-6-methylthio-4H-3,1-benzoxazin-4-one;

2-phenyloctyloxy-5-methyl-6,7-dichloro-4H-3,1-benzoxazin-4-one;

2-(1-phenyl-2-methyl-propyl)oxy-5-isopropoxy-4H-3,1-benzoxazin-4-one;

2-(1-phenyl-3-methyl-2-butyl)oxy-5-methyl-4H-3,1-benzoxazin-4-one;

2-(1-(4-isobutoxyphenyl)heptyloxy-5-methyl-7-methoxy-4H-3,1-benzoxazin-4-one

2-(4-ethylbenzyl)oxy-4H-3,1-benzoxazin-4-one;

2-(3,5-dimethoxybenzyl)oxy-4H-3,1-benzoxazin-4-one;

2-cyclopropyloxy-5-ethyl-4H-3,1-benzoxazin-4-one;

2-cyclobutyloxy-6-methylthio-4H-3,1-benzoxazin-4-one;

2-cyclohexyloxy-5-isopropyl-4H-3,1-benzoxazin-4-one;

2-(5-methylhexyloxy)-4H-3,1-benzoxazin-4-one;

2-(3-methyl-4-ethylpentyl)oxy-4H-3,1-benzoxazin-4-one;

2-(2-methylpentyl)oxy-7-nitro-4H-3,1-benzoxazin-4-one;

2-(1-[4-aminocyclohexyl]methyl)oxy-6-iodo-4H-3,1-benzoxazin-4-one;

2-(1-N,N-dimethylaminobenzyl)oxy-6-N,N-dimethylamino-4H-3,1-benzoxazin-4-one;

E. Similarly, the following nitro-substituted2-oxy-4H-3,1-benzoxazin-4-ones of Formula IA are prepared from thecorrespondingly substituted nitro-substituted anthranilic acids.(4-nitro-anthranilic acid is commercially available. Alternatively,these, as well as the 5- and 6-nitro anthranilic acids can be preparedaccording to the method set forth in Preparation I, above.):

2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one;

2-benzyloxy-7-nitro-4H-3,1-benzoxazin-4-one;

2-phenethyloxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-cyclopropyloxy-5-propyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-cyclobutyloxy-5-thiomethyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-cyclohexylyoxy-6-nitro-4H-3,1-benzoxazin-4-one;

2-phenylbutyloxy-7-nitro-4H-3,1-benzoxazin-4-one;

2-cyclopropylbutyloxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-(4-ethylbenzyl)oxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-(3,5-dimethoxybenzyl)oxy-5-iodo-7-nitro-4H-3,1-benzoxazin-4-one;

2-(4-thiomethylcyclohexyl)butyloxy-7-nitro-4H-3,1-benzoxazin-4-one;

2-methoxy-5-nitro-4H-3,1-benzoxazin-4-one;

6-nitro-2-propoxy-4H-3,1-benzoxazin-4-one;

5-methyl-7-nitro-2-pentoxy-4H-3,1-benzoxazin-4-one;

2-hexoxy-6-nitro-4H-3,1-benzoxazin-4-one;

8-nitro-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one;

2-isobutoxy-5-bromo-7-nitro-4H-3,1-benzoxazin-4-one;

EXAMPLE II A. Synthesis of 7-Amino-2-ethoxy-4H-3,1-benzoxazin-4-one, andRelated Compounds of Formula IB

7-Nitro-2-ethoxy-4H-3,1-benzoxazin-4-one (1 gm, 4.2 m mol.), 10% Pd-C (1gm), and cyclohexene (2.5 ml) were refluxed in dry benzene (50 ml) underanhydrous conditions for 3 hours. The hot reaction mixture was filteredimmediately through celite and the catalyst on celite was washed withhot benzene (25 ml). The combined filtrate was evaporated to drynessunder reduced pressure. The residue was stirred in anhydrous ether (30ml) to give 410 mg (47%) of the title compound,7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one, as a pale yellow powder, afterfiltration. The product can be further purified by chromatography onsilica gel column, if necessary (silica gel 60; EtOAc:pet. ether=1:2);m.p. 185°-187° C.; IR (KBr): ν_(max) 3425, 3330 cm⁻¹ (NH₂), 1740 cm⁻¹(C═O), 1640 cm⁻¹ (C═N); N'NMR (CDCl₃): δ 1.43 ppm (t, J=7.1 Hz, 3H, OCH₂CH₃), 4.30 ppm (b, 2H, NH₂), 4.75 ppm (q, J=7.1 Hz, 2H, OCH₂ CH₃),6.5-7.98 ppm (m, 3H, aromatic protons).

B. In a similar manner, but replacing the2-ethoxy-7-nitro-benzoxazin-4-one with other correspondingnitro-substituted compounds of Formula IA, (which are prepared asdescribed in Example I, paragraphs D and E, above), and followingcompounds of Formula IB are prepared:

7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one;

7-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one;

8-amino-2-ethoxy-4H-3,1-benzoxazin-4-one;

6-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one;

2-cyclopropyloxy-6,8-diamino-4H-3,1-benzoxazin-4-one;

5-amino-2-cyclobutyloxy-7-thiomethyl-4H-3,1-benzoxazin-4-one;

6-amino-2-cyclohexyloxy-4H-3,1-benzoxazin-4-one;

7-amino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one;

7-amino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one;

5-amino-2-methoxy-4H-3,1-benzoxazin-4-one;

6-amino-2-propoxy-4H-3,1-benzoxazin-4-one;

6,7-diamino-2-butoxy-4H-3,1-benzoxazin-4-one;

7-amino-5-methyl-2-pentoxy-4H-3,1-benzoxazin-4-one;

7-amino-2-hexoxy-4H-3,1-benzoxazin-4-one;

8-amino-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one; and

7-amino-2-isobutoxy-6-chloro-4H-3,1-benzoxazin-4-one;

7-ethyl-7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one

5-propyl-7-amino-2-benzyloxy-4H-3,1-benzoxazin-4-one

C. In a similar manner, but replacing the2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one with2-ethoxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one the following compoundof Formula IB was prepared:

2-ethoxy-5-ethyl-7-amino-4H-3,-benzoxazin-4-one, IR 3440, 3360, 3240,1735, 1660, 1640, 1610 cm

D. Similarly, but starting with other corresponding5-alkyl-7-nitro-substituted compounds of Formula IA, (which are preparedas described in Example XII, Paragraphs A, B and C), the followingcompounds of Formula IB are prepared:

2-ethoxy-5-methyl-7-amino-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-propyl-7-amino-4H-3,1-benzoxazin-4-one

2-ethoxy-5-isopropyl-7-amino-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-butyl-7-amino-4H-3,1-benzoxazin-4-one; and

2-ethoxy-5-isobutyl-7-amino-4H-benzoxazin-4-one.

EXAMPLE III A. 7-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one, andRelated Compounds of Formula IC

7-Amino-2-ethoxy-4H-3,1-benzoxazin-4-one (0.48 m mol., 100 mg) wasstirred in acetic anhydride (5 ml) at room temperature under anhydrousconditions for 30 minutes. The excess acetic anhydride was removed underreduced pressure at 35° C. (bath temperature). The residue was dissolvedin EtOAc (20 ml) and washed with saturated sodium bicarbonate solution(15 ml), water (15 ml). The organic layer was dried (MgSO₄) andevaporated to give the crude product. Chromatography on silica gel 60(EtOAc:pet. ether=3:2) gave 85 mg (71%) of the title compound,7-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one as a white powder; m.p.245°-246° C.; IR (KRr): ν_(max) 3360 cm⁻¹ (NH), 1740 cm⁻¹ (C═O), 1620cm⁻¹ (C═N), 1590 cm⁻¹ (aromatic amide); H'NMR (CDCl₃): δ1.44 ppm (t,J=711 Hz, 3H, OCH₂ CH₃), 2.24 ppm (s, 3H, CH₃ CO), 4.51 ppm (q, J=7.1Hz, 2H, OCH₂ CH₃), 7.30 ppm (b, 1H, NH), 7.28-8.10 ppm (m, 3H, aromaticprotons).

B. In a similar manner, but substituting other appropriate acidanhydrides (which are commercially available, or can be prepared as setforth in Preparation III, above) for the acetic anhydride, and othercorresponding nitro-substituted compounds of Formula IB (prepared asdescribed in Example I, paragraph E) the following compounds of FormulaIC are prepared:

5-acetylamino-2-ethoxy-4H-3,1-benzoxazin-4-one;

5-acryloylamino-2-ethoxy-4H-3,1-benzoxazin-4-one;

6-acetylamino-2-benzyloxy-4H-3,1-benzoxazin-4-one;

2-benzyloxy-6-butyrylamino-4H-3,1-benzoxazin-4-one;

5-crotonoylamino-2-cyclobutyloxy-7-thiomethyl-4H-3,1-benzoxazin-4-one;

6-acetylamino-2-cyclohexylyoxy-4H-3,1-benzoxazin-4-one;

2-cyclohexylyoxy-6-hexanoylamino-4H-3,1-benzoxazin-4-one;

7-acetylamino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one;

8-acetylamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

8-acryloylamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

7-acetylamino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one;

5-acetylamino-2-methoxy-4H-3,1-benzoxazin-4-one;

6-acetylamino-2-propoxy-4H-3,1-benzoxazin-4-one;

6-butyrylamino-2-propoxy-4H-3,1-benzoxazin-4-one;

7-acetylamino-5-methyl-2-pentoxy-4H-3,1-benzoxazin-4-one;

6-acetylamino-2-hexoxy-4H-3,1-benzoxazin-4-one;

8-acetylamino-2-(3-methylpentoxy)-4H-3,1-benzoxazin-4-one;

EXAMPLE IV A. 6-Carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one andRelated Compounds of Formula ID

To a solution of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one (0.2 m mol.,41 mg) and dry pyridine (0.1 ml) in dry dichloromethane (4 ml) at roomtemperature under anhydrous conditions was added ethyl chloroformate(0.05 ml, 0.52 m mol.) in one portion. After stirring for one hour, thereaction mixture was diluted with dichloromethane (16 ml), washedsuccessively with water (15 ml), 5% CuSO₄) and evaporated to dryness.The residue was stirred in anhydrous ether (2 ml) and filtered to afford53 mg (95%) of the title compound,7-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one as a colorlesspowder; m.p. 191°-193° C.; IR (KBr): ν_(max) 3310 cm⁻¹ (NH), 1735 cm⁻¹(ester, carbamyl); 1640 cm⁻¹ (C═N); H'NMR (CDCl₃): δ1.34 ppm (t, J=7.1Hz, 3H, CH₃ CH₂ OCON), 1.44 ppm (t, J=7.1 Hz, 3H, CH)

B. In a similar manner, but replacing the2-ethoxy-7-nitro-4H-3,1-benzoxazin-4-one with other 7-nitro-substitutedcompounds of Formula IA (which can be prepared according to the methodof Example I, paragraph E,) and substituting other suitable acid halidesof Formula III for the chloroformate, the following compounds of FormulaID are prepared:

5-carbomethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one;

5-carboethoxyamino-2-ethoxy-4H-3,1-benzoxazin-4-one;

2-benzyloxy-6-carboethoxyamino-4H-3,1-benzoxazin-4-one;

2-benzyloxy-6-carbopropoxyamino-4H-3,1-benzoxazin-4-one;

6-carbobutoxyamino-2-cyclohexyloxy-4H-3,1-benzoxazin-4-one;

6-carboethoxyamino-2-cyclohexylyoxy-4H-3,1-benzoxazin-4-one;

7-carbomethoxyamino-2-phenylbutyloxy-4H-3,1-benzoxazin-4-one;

8-carboethoxyamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

8-carbopropoxyamino-2-cyclopropylbutyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

7-carboethoxyamino-2-(4-thiomethylcyclohexyl)butyloxy-4H-3,1-benzoxazin-4-one;

5-carbomethoxyamino-2-methoxy-4H-3,1-benzoxazin-4-one;

6-carboethoxyamino-2-propoxy-4H-3,1-benzoxazin-4-one;

6-carbomethoxyamino-2-propoxy-4H-3,1-benzoxazin-4-one.

EXAMPLE V A. 7-(3-Diethylureido)-2-ethoxy-4H-3,1-benzoxazin-4-one andRelated Compounds of Formula IE

Trichloromethyl chloroformate (0.1 m mol., 13.3 μl) was added to asolution of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one (21 mg, 0.1 m mol)in dry THF (3 ml) at room temperature under argon. After stirring for 30minutes dry pyridine (0.1 ml) and diethylamine (0.1 ml) were added tothe reaction mixture and the stirring was continued for an additional 15minutes. The mixure was diluted with dichloromethane (20 ml) and washedsuccessively with water (15 ml), 5% CuSO₄ solution (2×10 ml), water (15ml). The organic layer was dried (MgSO₄) and evaporated to give asemi-solid residue. The residue was stirred in anhydrous ether (2 ml)and filtered to afford 12 mg (39%) of the expected carbamate derivative;m.p. 163°-164.5° C. IR (KBr): ν_(max) 3380 cm⁻¹ (NH), 1740 and 1730 cm⁻¹(C═O), 1670 cm⁻¹ (urea), 1640 cm⁻¹ (C--N); H'NMR (CDCl₃): 1.26 ppm (t,J=7.2 Hz, 6H, (CH₃ CH₂)₂ N), 1.43 ppm (t, J=7.2 Hz, 3H, CH₃ CH₂ O), 3.41ppm (q, J=7.2 Hz, 4H, (CH₃ CH₂)₂ N), 4.50 ppm (q, J=7.2 Hz, 2H, CH₃ CH₂O), 6.59 ppm (b, 1H, NH), 7.29-8.06 ppm (m, 3H, aromatic protons).

B. In a similar manner, but starting with the same or otheramino-substituted compounds of Formulas IA and IB, and replacing theethylamine where appropriate with other alkylamines, the followingcompounds of Formula IE are prepared:

7-(3-dimethylureido)-2-ethoxy-4H-3,1-benzoxazin-4-one;

7-(3-methylureido)-5-methyl-2-ethoxy-4H-3,1-benzoxazin-4-one.

EXAMPLE VI A. Preparation of5-Bromomethyl-2-ethoxy-4H,3,1-benzoxazin-4-one and Related Compounds ofFormula IF₁

A solution of 2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one (330 mg),N-bromosuccinimide (340 mg) and AIBN (10 mg,2,2'-azobis-iso-butyronitrile) was refluxed for 21/2 hours. The solutionwas evaporated to dryness. The residue was purified by columnchromatography (silica gel) 10% ethyl acetate:petroleum ether 30-60).This afforded the title compound,5-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 112°-114° C.; IR:1760, 1630, 1590 cm⁻¹.

B. In a similar manner, but starting with other appropriate compounds ofFormula I in which an R' is lower alkyl, the following compounds ofFormula IF₁ are obtained:

5-bromoethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

6-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

7-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

8-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

5-(5-bromopentyl)-2-isobutoxy-4H-3,1-benzoxazin-4-one;

5-bromomethyl-2-(1-cyclopropylethyl)oxy-4H-3,1-benzoxazin-4-one;

7-(iodoethyl-2-imidazolyloxy-4H-3,1-benzoxazin-4-one;

7-amino-5-(3-chloropropyl)-2-benzyloxy-4H-3,1-benzoxazin-4-one; and

5-(2-bromobutyl)-2-ethoxy-4H-3,1-benzoxazin-4-one.

EXAMPLE VII A. Preparation of5-(Dibromomethyl)-2-ethoxy-4H,3,1,-benzoxazin-4-one and RelatedCompounds of Formula IF₂

Proceeding in the same manner as Example VI, a solution of2-ethoxy-5-methyl-4H,3,1-benzoxazin-4-one (2 gm), N-bromosuccinimide(4.05 gm) and AIBN (25 mg) in carbon tetrachloride was heated for 4hours at reflux. The solvent was evaporated and the residue was purifiedby column chromatography (20% ethyl acetate: petroleum ether) to give5-(dibromomethyl)-2-ethoxy-4H-3,1-benzoxazin-4-one, m.p. 98°-99° C.; IR:1750, 1640 cm⁻¹.

B. In in the same manner, but replacing the2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one with other compounds ofFormula I, the following compounds of Formula IF₂ are prepared:

6-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

7-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

8-dibromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one;

5-(5-dibromopentyl)-2-isobutoxy-4H-3,1-benzoxazin-4-one;

5-dibromomethyl-2-(1-cyclopropylethyl)oxy-4H-3,1-benzoxazin-4-one; and

5-(dibromobutyl)-2-ethoxy-4H-3,1-benzoxazin-4-one.

EXAMPLE VIII A. Preparation of 2-Ethoxy-5-ethyl-4H,3,1-benzoxazin-4-oneand Related Compounds of Formula IG₁

Methyl lithium (4.69 ml, 1.4M, Aldrich) was added to a suspension ofcuprous iodide (0.62 gm) in anhydrous ether under argon at -78° C. Thesolution was stirred at -25° C. for 25 min. This solution was added to asolution of 2-ethoxy-5-bromomethyl-4H-3,1-benzoxazin-4-one (200 mg) in15 ml anhydrous ether and 3 ml dry tetrahydrofuran at -60° C. Reactionwas monitored by TLC until reaction completion. The reaction wasquenched with saturated ammonium chloride solution and filtered. Thefiltrate was extracted in the usual manner. The ethereal layer waswashed with water, dried over magnesium sulphate and evaporated to givean oil. The material was purified by thick layer chromatography (R_(f)=0.75, 3-% ethyl acetate: petroleum ether), to give2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one, m.p. 89°-91° C., H NMR: 1.3,1.5 (2t, 6H, 2CH₃), 3.2 (q, 2H, CH₂), 4.5 (q, 2H, OCH₂ CH₃), 7.2 (m, 2H,ArH), 7.6 (t, 1H, ArH).

B. Proceeding in the same manner, but replacing the lithium dimethylcuprate, where desired, with other lithium dialkyl cuprates, andstarting with other appropriate compounds of Formula IF₁, the followingcompounds of Formula IG₁ are prepared:

2-ethoxy-5-propyl-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-butyl-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-pentyl-4H-3,1-benzoxazin-4-one;

2-benzyloxy-5-methyl-4H-3,1-benzoxazin-4-one;

2-phenethyloxy-6-ethyl-4H-3,1-benzoxazin-4-one;

2-cyclopropyloxy-5-n-propyl-4H-3,1-benzoxazin-4-one; and

2-(1-cyclohexylethyl)oxy-5-ethyl-3,1-benzoxazin-4-one.

EXAMPLE IX A. Preparation of1-(2-Ethoxy-4H-3,1-benzoxazin-4-on-5-yl)-methyl-triphenylphosphoniumbromide and Related Compounds of Formula IG₂

A solution of 5-bromomethyl-2-ethoxy-4H-3,1-benzoxazin-4-one (3.15 gm),prepared as described in Example VI above, and triphenylphosphine (5.44gm) in toluene was heated at 60° C. for 6 hours. The insolubleprecipitate was filtered, and the mother liquor was reduced to half ofits original volume and refiltered, yielding 9 gm of1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)-methyl-triphenylphosphoniumbromide, m.p. (turns yellow at) 125° C. IR: 1740, 1640 cm⁻¹ ;, decom.135°-140° C.

B. In a similar manner, but starting instead with other desiredcompounds of Formula IF₁, the preparation of which is described inExample VI, the following compounds of Formula IG₂ are prepared:

1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)ethyl-triphenylphosphoniumbromide;

1-(2-ethoxy-4H-3,1-benzoxazin-4-on-6-yl)methyl-triphenylphosphoniumbromide;

1-(2-ethoxy-4H-3,1-benzoxazin-4-on-7-yl)methyl-triphosphonium bromide;

1-(2-ethoxy-4H-3,1-benzoxazin-4-on-8-yl)methyl-triphenylphosphoniumbromide;

1-(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)butyl-triphenylphosphoniumbromide.

1-(2-isopropoxy-7-nitro-4H-3,1-benzoxazin-4-on-5-yl)ethyl-triphenylphosphoniumbromide;

1-[2-(4-methylcyclohexyl)oxy-4H-3,1-benzoxazin-4-on-6-yl]-methyl-triphenylphosphoniumbromide.

EXAMPLE X A. Preparation of(2-Ethoxy-1-propenyl)-4H-3,1-benzoxazin-4-one and Related Compounds ofFormula IG₃

To a well-stirred suspension of(2-ethoxy-4H-3,1-benzoxazin-4-on-5-yl)methyltriphenylphosphoniumbromide, prepared as described in Example IX, at -60° C. under argon,was added DBu (1-8-diazabicyclo[5,4,0]-undec-7-ene), 0.85 ml. After 30min. at -40° C., 2 ml of acetaldehyde was added. The solution wasstirred for 2 hours and warmed to room temperature. Solvent evaporationgave a residual oily solid which was chromatographed (silica gel 30%ethyl acetate:petroleum ether) to give the title compound,2-ethoxy-5-(propenyl)-4H-3,1-benzoxazin-4-one, (R_(f) =0.77); IR: 1620,1745 cm⁻¹.

B. Proceeding in a similar manner, but replacing acetaldehyde with otheralkylaldehydes, the following compounds of Formula IG₃ are prepared:

2-ethoxy-5-(1-butenyl)-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-(1-hexenyl)-4H-3,1-benzoxazin-4-one;

2-ethoxy-5-(1-pentenyl)-4H-3,1-benzoxazin-4-one

2-methoxy-5-(1-butenyl)-4H-3,1-benzoxazin-4-one;

2-n-propoxy-5-(1-propenyl)-7-methylamino-4H-3,1-benzoxazin-4-one;

2-ethoxy-6-(1-butenyl)-4H-3,1-benzoxazin-4-one;

5-(1-hexenyl)-2-isopropoxy-7-nitro-4H-3,1-benzoxazin-4-one;

6-(1-butenyl)-2-(4-methylcyclohexyl)oxy-4H-3,1-benzoxazin-4-one;

2-n-butoxy-5-(isopropenyl)-7-methoxy-4H-3,1-benzoxazin-4-one;

2-s-butoxy-5-(isopentenyl)-4H-3,1-benzoxazin-4-one;

2-n-octyloxy-(5-pentenyl)-7-nitro-4H-3,1-benzoxazin-4-one.

EXAMPLE XI Compounds of Formula IH A. Preparation of Methyl4,5-dimethoxy-2-[4-(N-triphenylmethyl)-imidazolyl)carbomethyloxy]amino-benzoateand Related compounds of Formula VI

Trichloromethyl chloroformate (0.16 ml, 1.2 mmol.) was added to asolution of methyl 6,7-dimethoxyanthranilate (422 mg, 2 mmol.) in dryTHF (30 ml) at room temperature under argon. After stirring for 90minutes, anhydrous triethylamine (2 ml, 14.4 mmol.) and4(N-triphenylmethyl)imidazolymethanol (749 mg, 2.2 mmol.) were added andthe mixture was refluxed for one hour. The solvent was removed underreduced pressure. The residue was shaken with a mixture of ether/water(40 ml/40 ml) and the insoluble solid was collected by filtration toafford 936 mg (81%) of methyl4,5-dimethoxy-2-[4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]aminobenzoate as a fine powder; m.p. 190°-192° C.; IR (KBr): ν_(max) 32670cm⁻¹ (NH), 1730 cm⁻¹ (carbamate), 1690 (COOCH₃); H'NMR (CDCl₃): δ3.88ppm (s, 6H, OCH₃), 5.13 ppm (s, 2H, OCH₂), 6.95-7.44 ppm (m, 18H,aromatic protons and imidazolyl C --H), 8.16 (s, 1H, imidazolyl C₂ --H).10.49 ppm (s, 1H, NH).

B. In like manner, but starting instead with other appropriatelysubstituted anthranilates, the following representative compounds ofFormula VI are obtained:

methyl6-methyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate;

methyl 6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate;

methyl6-chloro-4-nitro-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate;

methyl6-ethylthio-4-bromomethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate;

methyl4-amino-6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate, and

methyl4-amino-6-ethyl-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]-aminobenzoate.

C. Preparation of6,7-Dimethoxy-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-oneand Related Compounds of Formula IH

Methyl4,5-dimethoxy-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]amino-benzoate(400 mg, 0.72 mmol.), prepared as described in Paragraph A of thisExample, was stirred in a solution of 1N NaOH (10 ml), THF (20 ml), andmethanol (20 ml) at room temperature for 3 hours. The organic solventwas removed under reduced pressure at 35° C. (bath temperature). Theaqueous residue was diluted with water (15 ml) and acidified to pH 4with 1N HCl. The white precipitate was collected by filtration to yield73 mg (18%) of the expected product. The acidic filtrate was saturatedwith sodium chloride and extracted with ethyl acetate (2×20 ml). Thecombined ethyl acetate extract was droed (MgSO₄), evaporated to dryness.The residue was stirred in anhydrous ether (5 ml) and filtered to afford173 mg (42%) of4,5-dimethoxy-2-[(4-(N-triphenylmethyl)imidazolyl)carbomethyloxy]aminobenzoicacid. The combined yield of the expected acid was 246 mg (60%); mp. 200°C. (dec); IR (KBr): ν_(max) 3600-2800 cm⁻¹ (b, COOH), 1730 cm⁻¹(carbamate), 1670 cm⁻¹ (COOH); H'NMR (DMSO-d₆): δ3.85 ppm (s, 6H,(OCH₂), 6.85-7.65 ppm (m, 18H, aromatic protons and imidazolyl C₅ --H),7.90 (s, 1H, imidazolyl C₂ --H), 11.07 ppm (b, 1H, NH).

1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (21 mg, 0.11mmol.) was added in one portion to a suspension of the4,5-dimethoxy-2-[(4-triphenylmethyl)imidazolyl)carbomethyloxy]aminobenzoicacid (49 mg, 0.09 mmol.) in anhydrous dichloromethane (2.5 ml) at roomtemperature under anhyrous conditions. After stirring for 30 minutes theclear reaction mixture was diluted with dichloromethane (17.5 ml) andwashed with water (2×20 ml). The organic layer was dried with magnesiumsulfate and evaporated to give a colorless syrup which was trituratedwith 50% pentane in anhydrous ether (3 ml) to give 35 mg (71%) of6,7-dimethoxy-2-(4(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-oneas a white powder; m.p. 130°-133° C.; IR (KBr): ν_(max) 11760 cm⁻¹(C═O), 1630 cm⁻¹ (C═N).

D. In a similar manner, the following representative compounds ofFormula IH are prepared from the corresponding amino-benzoates ofFormula VI, the preparation of which is described in Paragrapghs A and Bof this Example:

6-methyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-one;

6-ethyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-one;

6-ethylthio-4-bromomethyl-2-(4-(N-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-one;and

7-amino-5-ethyl-2-(4-(4-triphenylmethyl)imidazolyl)methyloxy-4H-3,1-benzoxazin-4-one.

EXAMPLE XII A. Preparation of2-ethoxy-5-ethyl-7-nitro-H-3,1-benzoxazin-4-one and Related Compounds ofFormula IA

A solution of 2-carboethoxyamino-4-nitro-6-ethyl-benzoic acid and1-(3-dimethylaminopropyl)-3-ethylcarbodimide hydrochloride in anhydrousTHF (25 ml) was stirred at room temperature for 21/2 hrs. The solutionwas evaporated by dryness and the residue partitioned between ethylacetate and water. The ethyl acetate layer was dried over magnesiumsulphate and evaporated to a solid. The solid was recrystallized frommethylene chloride: petroleum ether to give the title compound,2-ethoxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one, m.p. 106°-107° C., Ir.1770, 1660, 1600, 1595, 1535, 1515 cm.

B. In a similar manner, the following representative compounds ofFormula IA are prepared from the corresponding amino-benzoic acids ofFormula VII, the preparation of which is described in Preparation VI:

2-ethoxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-benzyloxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-isopropyloxy-5-propyl-7-nitro-4H-3,1-benzoxazin-4-one;

2-isopropyloxy-5-butyl-4-nitro-4H-3,1-benzoxazin-4-one; and

2-cyclopropyloxy-5-iso-butyl-4-nitro-4H-3,1-benzoxazin-4-one.

EXAMPLE XIII Conversion of Free Base to Acid Addition Salt

A stoichiometric amount of 3% hydrogen chloride in dioxane is added to asolution of 1.0 g. of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one in 20 mldioxane. Diethyl ether is added until precipitation is complete. Theproduct is filtered, washed with ether, air dried and recrystallized togive 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one hydrochloride.

In a similar manner, other compounds of Formula I in free base form maybe converted to the acid addition salts by treatment with theappropriate acid, for example, hydrochloric acid, hydrobromic acid,sulfuric acid, nitric acid, phosphoric acid, acetic acid, propionicacid, glycolic acid, pyruvic acid, oxalic acid, malonic acid, succinicacid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,ethanesulfonic acid, p-toluenesulfonic acid, and the like.

EXAMPLE XIV Conversion of Salt to Free Base

1.0 g of 7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one HCl suspended in 50 mlof ether is stirred with a twofold stoichiometric excess of diluteaqueous potassium carbonate solution until the salt is completelydissolved. The organic layer is then separated, washed twice with water,dried over magnesium sulfate and evaporated to yield7-amino-2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one as the free base.

EXAMPLE XV Direct Interchange of Acid Addition Salts

7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one acetate (1.0 g) is dissolved in50 ml water containing a stoichiometric equivalent of sulfuric acid, andthe solution evaporated to dryness. The product is suspended in etherand filtered, air dried and recrystallized from methanol/acetone toyield 2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one sulfate.

In Examples XIII through XX, the active ingredient is7-amino-2-ethoxy-4H-3,1-benzoxazin-4-one. Other compounds of Formula Iand the pharmaceutically acceptable salts thereof may, of course, besubstituted.

EXAMPLE XVI

    ______________________________________                                                         Quantity per                                                 Ingredients      tablet, mgs.                                                 ______________________________________                                        Active ingredient                                                                              25                                                           cornstarch       20                                                           lactose, spray-dried                                                                           153                                                          magnesium stearate                                                                              2                                                           ______________________________________                                    

The above ingredients are thoroughly mixed and pressed into singlescored tablets.

EXAMPLE XVII

    ______________________________________                                                         Quantity per                                                 Ingredients      tablet, mgs.                                                 ______________________________________                                        Active ingredient                                                                              100                                                          lactose, spray-dried                                                                           148                                                          magnesium stearate                                                                              2                                                           ______________________________________                                    

The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

EXAMPLE XVIII

    ______________________________________                                                         Quantity per                                                 Ingredients      tablet, mgs.                                                 ______________________________________                                        Active ingredient                                                                              200                                                          cornstarch        50                                                          lactose          145                                                          magnesium stearate                                                                              5                                                           ______________________________________                                    

The above ingredients are mixed intimately and pressed into singlescored tablets.

EXAMPLE XIX

    ______________________________________                                                         Quantity per                                                 Ingredients      tablet, mgs.                                                 ______________________________________                                        Active ingredient                                                                              108                                                          lactose          15                                                           cornstarch       25                                                           magnesium stearate                                                                              2                                                           ______________________________________                                    

The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

EXAMPLE XX

    ______________________________________                                                         Quantity per                                                 Ingredients      tablet, mgs.                                                 ______________________________________                                        Active ingredient                                                                              150                                                          lactose           92                                                          ______________________________________                                    

The above ingredients are mixed and introduced into a hard-shell gelatincapsule.

EXAMPLE XXI

An injectable preparation buffered to a pH of 7 is prepared having thefollowing composition:

    ______________________________________                                        Ingredients                                                                   ______________________________________                                        Active ingredient      0.2       g                                            KH.sub.2 PO.sub.4 buffer (0.4 M solution)                                                            2         ml                                           KOH (1 N)              q.s. to pH 7                                           water (distilled, sterile)                                                                           q.s. to 20                                                                              ml                                           ______________________________________                                    

EXAMPLE XXII

An oral suspension is prepared having the following composition:

    ______________________________________                                        Ingredients                                                                   ______________________________________                                        Active ingredient     0.1       g                                             fumaric acid          0.5       g                                             sodium chloride       2.0       g                                             methyl paraben        0.1       g                                             granulated sugar      25.5      g                                             sorbitol (70% solution)                                                                             12.85     g                                             Veegum K (Vanderbilt Co.)                                                                           1.0       g                                             flavoring             0.035     ml                                            colorings             0.5       mg                                            distilled water       q.s. to 100                                                                             ml                                            ______________________________________                                    

EXAMPLE XXIII Topical Formulation

    ______________________________________                                        Ingredients           grams                                                   ______________________________________                                        Active compound       0.2-2                                                   Span 60               2                                                       Tween 60              2                                                       Mineral oil           5                                                       Petrolatum            10                                                      Methyl paraben        0.15                                                    Propyl paraben        0.05                                                    BHA (butylated hydroxy anisole)                                                                     0.01                                                    Water                 q.s. 100                                                ______________________________________                                    

All of the above ingredients, except water, are combined and heated to60° C. with stirring. A sufficient quantity of water at 60° C. is thenadded with vigorous stirring to emulsify the ingredients, and water thenadded q.s. 100 g.

EXAMPLE XXIV Human Leukocyte Elastase Inhibition Assay 1. Enzyme

References:

Barrett, A. J. (1981), Methods in Enzymology, 80C, 581-588.

Engelbrecht, et al., (1982), Z. Physiol. Chem., 363, 305-315.

Fresh human leukocytes were obtained from a healthy donor, frozen andkept at -75° C. until use. Enzyme preparation followed the abovereferenced methods: cells were washed in saline, homogenized in thepresence of 1M NaCl and 0.1% Brij 35 (Sigma Chemical C., No. P-1254).After centrifugation and concentration by dialysis against polyethyleneglycol (MW 20,000), the material was chromatographed on Sephacryl S-300(Pharmacia). Active fractions were combined, concentrated as before, andchromatographed on an affinity gel of bovine lung trypsin inhibitorattached to Sepharose CL-4B. Active fractions were combined,concentrated as before to approximately 0.3 micromolar in activeelastase, and frozen in 1 ml aliquots at -75° C. until use.

2. Substrate

Methoxysuccinyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-N-methyl-coumarinamidewas obtained from Peninsula Laboratories, San Carlos, Calif. Solutionsof 1 mM in dimethylsulfoxide were made and kept at 4° C. until use.

3. Inhibitors

The compounds of Formula I to be assayed were dissolved indimethylsulfoxide to give 5, 10, or 20 mM stock solutions, which may befurther diluted as required.

4. Assay Buffer

The buffer consisted of 25 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid, 1M sodium chloride, 0.1% w/v Brij 35, pH 7.8.

5. Procedure

A Perkin-Elmer Model 650-40 fluorescence spectrophotometer is set up asfollows: ratio mode, excitation 370 nm, emission 460 nm, full scaleoutput 1, 5, or 10 units, cell compartment thermostatted at 25° C. Forthose compounds of Formula I which are themselves fluorescent, theexcitation wavelength may be optionally 390 nm to minimize interference.To 2.0 ml of assay buffer in a fluorescence cuvette is added 5microliters substrate and 20 microliters enzyme, with mixing. The changein fluorescence is recorded on a strip chart recorder to measure theinitial, uninhibited rate, typically 0.8 units per minute. Afterapproximately two minutes of such recording, inhibitor (between 0.5 and20 microliters of the stock solution) is added with mixing, andrecording continued. The reaction is recorded until a new constant rateis achieved. This procedure is repeated for several (4-6) inhibitorconcentrations. The data--a table of substrate concentration, inhibitorconcentration, and observed reaction velocities--are fit to theappropriate equation by non-linear least squares multiple regression.

EXAMPLE XXV Human Thrombin Inhibition Assay 1. Enzyme

Human thrombin number T-8885 was obtained from Sigma Chemical Company,St. Louis, Mo., and reconstituted with water to approximately 2.5 NIHunits/ml.

2. Substrate

BOC-L-Valyl-L-prolyl-L-arginyl-N-methylcoumarinamide was obtained fromPeninsula Laboratories, San Carlos, Calif. Solutions were made to 1 mMin dimethyl sulfoxide.

3. Inhibitors

As Example XXI.

4. Assay Buffer

The assay buffer consisted of 25 mM N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 0.5M sodium chloride, 0.1% w/vpolyethylene glycol 8000, pH 7.8.

5. Procedure

The procedure was as in Example XXI, except that 5 microliters ofsubstrate and 2.5 microliters enzyme solution were used.

EXAMPLE XXVI Human Urokinase Inhibition Assay 1. Enzyme

Human Urokinase was obtained from Leo Laboratories, Pickering, Ontario,and made to approximately 2.5 mg/ml in 0.10M sodium citrate, 50 mMsodium chloride, pH 3.

2. Substrate

Glutaryl-glycyl-L-arginyl-methyl coumarin amide (Peninsula Laboratories,vide supra) was made to approximately 1 mM in 1:1water:dimethylsulfoxide.

3. Inhibitors

As Example XXI.

4. Assay Buffer

The assay buffer consisted of 50 mM tris(hydroxymethyl)amino methane,0.10M sodium chloride, 10 mM calcium chloride, pH 8.0.

5. Procedure

The procedure was as in Example XXI, with 5 microliters enzyme used.

EXAMPLE XXVII Bovine Chymotrypsin Inhibition Assay 1. Enzyme

Chymotrypsin type II was obtained from Sigma Chemical Company and madeto 0.25 mg/ml in 1 mM hydrochloric acid and kept at 4° C. until use.

2. Substrate

7-(Glutaryl-L-phenylalaninamido)-4-methyl coumarin was obtained fromSigma and made to 10 mM in 1:1 acetonitrile:dimethylsulfoxide.

3. Inhibitors

As Example XXI.

4. Assay Buffer

The assay buffer consisted of 25 mM N-2-hydroxy ethylpiperazine-N-2-ethane sulfonic acid, 0.1M potassium chloride, pH 7.8.

5. Procedure

As Example XXI.

EXAMPLE XXVIII Boar Acrosin Inhibition Assay

1. Enzyme

Boar acrosin was a gift of Professor W. Muller-Esterl, as purified inMuller-Esterl, et al., Hoppe-Seyler's Z. Physiol. Chem., 361, 1811-1821,1980, and was made to approximately 0.1 mg/ml in 1 mM HCl and kept at 4°C. until use.

2. Substrate

7-(N-benzoyl-L-argininamido)-4-methyl coumarin HCl was obtained fromSigma Chemical Company and made up to 2 mM in dimethyl sulfoxide.

3. Inhibitors

As Example XXI.

4. Assay Buffer

The assay buffer consisted of 0.10M N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid, 50 mM calcium chloride, 0.01%v/v Triton X-100, pH 7.8.

5. Procedure

The procedure was as Example XXI, with 5 microliters substrate and 2.5to 15 microliters enzyme as required to obtain approximately 0.5fluorescent unit/minute uninhibited rate.

EXAMPLE XXIX Assay for Stability of Compounds in Whole Plasma

Whole, citrated human plasma was obtained from a local blook bank andkept frozen at -70° C. until use. Benzoxazinone (from a 10 mM stocksolution in dimethylsulfoxide) was added to plasma at 37° C. to a finalconcentration of 50 mM, and incubation was continued at 37°. At varioustimes thereafter, aliquots were withdrawn and diluted 5-fold into 20 mMpotassium phosphate, 0.14M sodium chloride, 3% w/v Brij 35 (SigmaChemical Company), pH 7.4, and the fluorescence of this solution wasmonitored at 345 nm (excitation) and 429 nm (emission). The fluorescenceintensity is proportional to the concentration of benzoaxazinoneremaining. These data were fit by interative non-linear techniques tofirst-order exponentials to obtain the half-times in plasma.

Alternatively, for benzoxazininones which are weakly or non-fluorescent,high pressure liquid chromatography (HPLC) was used. From plasmaincubations as above, aliquots were withdrawn and diluted 1:1 (v/v) withacetonitrile, mixed on a vortex stirrer, and centrifuged. Tenmicroliters of the supernatant was injected into the HPLC andchromatographed on a 5 micron RP-18 (reverse phase) column, in 9%acetonitrile, 10% water (v/v), with detection by absorbance at 340 nm.Retention times and concentrations were determined by comparison tostandards. The integrated areas of the benzoxazinone peaks vs.incubation time were treated as above to obtain half-times.

EXAMPLE XXX Assay for Inhibition of Endothelial Cell Basement MembraneDegredation Principle

Various serine proteases, including elastase, are secreted by murineactivated macrophages, and cause the degredation of endothelial cellbasement membrane. This assay tests the inhibitory potency of a testcompound to as a measure of its ability to inhibit enzyme induceddegredation of the endothelial cell basement membrane. PG,86

Methods

LE II murine lung capillary endothelial cells were grown to confluencein 24 well cluster dishes. Cells were lageled with 10 μCi [³⁵S]-methionine per well, in medium deficient in methionine andsupplemented with 10% fetal calf serum and 10 μg/ml ascorbate for twodays.

Biosynthetically labeled basement membranes were prepared by lysing thecells with 0.5% Nonidet P40 for 5 minutes at room temperature andremoving cytoskeletal degris with 0.25N NH₄ OH, followed by severalwashes with phosphate buffer.

Activate murine macrophages were obtained by peritoneal lavage of femaleC3H/He mice injected intraperitoneally with corynebacterium parvum 7days prior to the experiment.

Macrophages were either layere on the basement membrane (5×10⁵cells/well) or used to prepare conditioned medium in separate dishesthat were then incubated with the basement membrane.

Test compound was dissolved in DMSO at 10⁻² M-10⁻³ M, diluted withmedium containing 1 mg/ml bovine serum albumin, and tested at 10⁻⁵ M.

Machrophages, conditioned medium, or 5 μ/ml porcine pancreatic elastase(Sigma) were incubated with the basement membrane at 37° C. for 4 hoursin the presence or absence of test compound. Aliquots of the medium werecounted for ³ H and ³⁵ S in a liquid scintillation counter.

Results may be expressed as % inhibition of the release of radioactivityafter correction for spontaneous release of radioactivity determined incontrol wells. Compounds of Formula I which were tested in this assayexhibited significant inhibitory potency against degredation of thebasement membrane.

What we claim is:
 1. A compound of the formula: ##STR33## wherein, a isan integer of 0-4;A is a bond, or alkylene having one to eight carbonatoms; R is phenyl, imidazolyl, or cycloalkyl having three to six carbonatoms, wherein the phenyl or cycloalkyl ring is optionally substitutedwith 1-3 substituents independently selected from the group consistingof lower alkyl having one to four carbon atoms, lower alkoxy having oneto four carbon atoms, --N(R¹)₂, --NO₂, halo, and lower thioalkyl havingone to four carbon atoms, and each R' is independently selected from thegroup consisting of lower alkyl having one to six atoms, lower alkenylhaving two to six carbon atoms, lower alkoxy having one to six carbonatoms, NO₂, halo-lower alkyl or lower alkylthio having one to six carbonatoms, halo, --N(R¹)₂, ##STR34## --NR¹ COR² and ##STR35## in which eachR¹ is independently hydrogen or lower alkyl having one to six carbonatoms, or together form a piperidine or piperazine ring optionallysubstituted at the ring nitrogen by lower alkyl having one to fourcarbon atoms or --CH₂ CH₂ OH, andeach R² is independently lower alkylhaving one to four carbon atoms,or a pharmaceutically acceptable acidaddition salt thereof.
 2. A pharmaceutical composition which comprises atherapeutically effective amount of a compound of claim 1, or apharmaceutically acceptable acid addition salt thereof, in admixturewith at least one pharmaceutically acceptable excipient.
 3. A compoundof claim 1 in which A is alkylene having one to four carbon atoms.
 4. Acompound of claim 3 in which A is methylene.
 5. A compound of claim 1 inwhich a is at least one.
 6. A compound of claim 5 in which an R' is inthe 5-position.
 7. A compound of claim 6 in which the R' in the5-position is lower alkyl having one to three carbon atoms.
 8. Acompound of claim 5 in which an R' is in the 7-position.
 9. A compoundof claim 8 in which A is methylene, R is phenyl, a is 2, and R' ismethoxy in the 6- and 7-positions, namely2-benzyloxy-6,7-dimethoxy-4H-3,1-benzoxazin-4-one, or a pharmaceuticallyacceptable acid addition salt thereof.
 10. A compound of claim 6 inwhich an R' is in the 7-position.
 11. A compound of claim 10 in whichthe R' at the 5-position is lower alkyl having one to six carbon atomsor lower alkenyl having two to six carbon atoms, and the R' at the7-position is selected from the group consisting of lower alkoxy havingone to six carbon atoms, --N(R¹)₂, ##STR36## --NR¹ COR² and ##STR37##12. A compound of claim 11 in which the R' in the 5-position is loweralkyl having one to three carbon atoms and the R' in 7-position is--N(R¹)₂ or --NR¹ COOR² in which each R¹ is independently hydrogen,methyl or ethyl and R² is methyl or ethyl.
 13. A compound of theformula: ##STR38## wherein: a is an integer of 1 to 4;A is alkylenehaving one to eight carbon atoms; R is hydrogen; andat least one R'represents a substituent at one of the 5- and 7-positions, wherein saidsubstituent is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkoxy having one to sixcarbon atoms, lower alkenyl having two to six carbon atoms, halo-loweralkyl having one to six carbon atoms, lower alkylthio having one to sixcarbon atoms, halo, --NO₂, 'N(R¹)₂, ##STR39## --NR¹ COR², and ##STR40##in which each R¹ is independently hydrogen or lower alkyl having one tofour carbon atoms, or together form a piperidine or piperazine ringoptionally substituted at the ring nitrogen by lower alkyl having one tofour carbon atoms or --CH₂ CH₂ OH, and each R² is independently loweralkyl having one to four carbon atoms,or a pharmaceutically acceptableacid addition salt thereof.
 14. A compound of claim 13 in which A isalkylene having one to four carbon atoms.
 15. A compound of claim 14 inwhich A is methylene or ethylene.
 16. A compound of claim 15 in which anR' is in the 5-position.
 17. A compound of claim 13 in which an R' is inthe 7-position.
 18. A compound of claim 17 in which A is ethylene ormethylene.
 19. A compound of claim 18 in which R' is selected from thegroup consisting of --NO₂, --N(R¹)₂, ##STR41## --NR¹ COR², and ##STR42##20. A compound of claim 16 in which an R' is in the 7-position.
 21. Acompound of claim 16 in which A is ethylene, a is 1 and R' is ethyl,namely 2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one.
 22. A compound of claim16 in which A is ethylene, a is 1 and R' is methyl, namely2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one.
 23. A compound of claim 16 inwhich A is ethylene, a is 1 and R' is n-propyl, namely2-ethoxy-5-n-propyl-4H-3,1-benzoxazin-4-one.
 24. A compound of claim 16in which A is ethylene, a is 1 and R' is isopropyl, namely2-ethoxy-5-isopropyl-4H-3,1-benzoxazin-4-one.
 25. A compound of claim 20in which the R' in the 5-position is lower alkyl havng one to six carbonatoms or lower alkenyl having two to six carbon atoms, and the R' in the7-position is selected from the group consisting of lower alkoxy havingone to six carbon atoms, --N(R¹)₂, ##STR43## --NR¹ COR² and ##STR44##26. A compound of claim 25 in which the R' in the 5-position is loweralkyl having one to three carbon atoms and the R' in the 7-position is--N(R¹)₂ or --NR¹ COOR² in which each R¹ is independently hydrogen,methyl or ethyl and R² is methyl or ethyl.
 27. A compound of theformula: ##STR45## wherein: a is an integer of 2 to 4;A is alkylenehaving one to eight carbon atoms; R is hydrogen; anda first R'represents a substituent at the 5-position, and a second R' represents asubstituent at the 7-position, and each R' is independently selectedfrom the group consisting of lower alkyl having one to six carbon atoms,lower alkenyl having two to six carbon atoms, lower alkoxy having one tosix carbon atoms, halo, halo-lower alkyl having one to six carbon atoms,lower alkylthio having one to six carbon atoms, --NO₂, --N(R¹)₂,##STR46## --NR¹ COR², and ##STR47## in which each R¹ is independentlyhydrogen or lower alkyl having one to six carbon atoms, or together forma piperidine or piperazine ring optionally substituted at the ringnitrogen by lower alkyl having one to four carbon atoms or --CH₂ CH₂ OH,and each R² is independently lower alkyl having one to four carbonatoms,or a pharmaceutically acceptable acid addition salt thereof.
 28. Acompound of claim 27 in which an R' is in the 5-position.
 29. A compoundof claim 28 in which an R' is in the 7-position.
 30. A compound of claim28 in which the R' in the 5-position is lower alkyl having one to threecarbon atoms.
 31. A compound of claim 29 in which the R' in the5-position is lower alkyl or lower alkenyl and the R' in the 7-positionis selected from the group consisting of lower alkoxy, --N(R¹)₂,##STR48## --NR¹ COR², and ##STR49##
 32. A compound of claim 31 in whichthe R' in the 5-position is lower alkyl having one to three carbon atomsand the R' in the 7-position is --N(R¹)₂ or --NR¹ COOR¹ in which each R¹is independently hydrogen, methyl or ethyl and R² is methyl or ethyl.33. A compound of the formula: ##STR50## wherein: a is an integer of 0to 3;A is a bond, or alkylene having one to eight carbon atoms; R ishydrogen, imidazolyl, phenyl, or cycloalkyl having thre to six carbonatoms, wherein the phenyl, imidazolyl or cycloalkyl ring is optionallysubstituted with 1-3 substituents independently selected from the groupconsisting of lower alkyl having one to four carbon atoms, lower alkoxyhaving one to four carbon atoms, --N(R¹)₂, --NO₂, halo, and lowerthioalkyl having one to four carbon atoms; and each R' and R" areindependently selected from the group consisting of lower alkyl havingone to six carbon atoms, lower alkenyl having two to six carbon atoms,halo, lower alkoxy having one to six carbon atoms, halo-lower alkylhaving one to six carbon atoms, lower alkylthio having one to six carbonatoms, --NO₂, --N(R¹)₂, --NR¹ COR², ##STR51## in which each R¹ isindependently hydrogen or lower alkyl having one to six carbon atoms, ortogether form a piperidine or piperazine ring optionally substituted atthe ring nitrogen with lower alkyl having one to four carbon atoms, or--CH₂ CH₂ OH;each R² is independently lower alkyl having one to fourcarbon atoms, and A is alkylene if R is hydrogen,or a pharmaceuticallyacceptable acid addition salt thereof.
 34. A compound of claim 33 inwhich A is alkylene of one to four carbon atoms.
 35. A compound of claim34 in which A is methylene or ethylene.
 36. A compound of claim 33 inwhich R" is lower alkyl having one to six carbon atoms or lower alkenylhaving two to six carbon atoms.
 37. A compound of claim 36 in which R"is lower alkyl having one to three carbon atoms.
 38. A compound of claim37 in which a is zero.
 39. A compound of claim 37 in which a is at leastone.
 40. A compound of claim 39 in which an R' is in the 7-position. 41.A compound of claim 40 in which the R' in the 7-position is selectedfrom the group consisting of lower alkoxy having one to six carbonatoms, --N(R¹)₂, ##STR52## --NR¹ COR² and ##STR53##
 42. A compound ofclaim 41 in which R" is lower alkyl having one to six carbon atoms orlower alkenyl having two to six carbon atoms.
 43. A compound of claim 42in which the R" is lower alkyl having one to three carbon atoms and theR' in the 7-position is --N(R¹)₂ or --NR¹ COOR¹ in which each R¹ isindependently hydrogen, methyl or ethyl and R² is methyl or ethyl.
 44. Acompound of the formula: ##STR54## wherein: a is 1 or 2 and representsthe number of R' substituents;A is a bond or alkylene having one toeight carbon atoms; R is hydrogen, phenyl, imidazolyl or cycloalkylhaving three to six carbon atoms, wherein the phenyl or cycloalkyl ringis optionally substituted with 1-3 substituents independently selectedfrom the group consisting of lower alkyl having one to four carbonatoms, lower alkoxy having one to four carbon atoms, --N(R¹)₂, --NO₂,halo, and lower thioalkyl having one to four carbon atoms; and each R'represents a substituent at one of the 5- or 7-positions, wherein saidsubstituent is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, lower alkoxy having one to six carbon atoms, halo, --NO₂,halo-lower alkyl having one to six carbon atoms, lower alkylthio havingone to six carbon atoms, --N(R¹)₂, ##STR55## --NR¹ COR², and ##STR56##in which each R¹ is independently hydrogen or lower alkyl having one tosix carbon atoms, or together form a piperidine or piperazine ringoptionally substituted at the ring nitrogen with lower alkyl having oneto four carbon atoms or --CH₂ CH₂ OH;each R² is independently loweralkyl having one to four carbon atoms and A is alkylene if R ishydrogen,or a pharmaceutically acceptable acid addition salt thereof.45. A compound of claim 44 in which A is alkylene having one to eightcarbon atoms.
 46. A compound of claim 45 in which a is 1 and R' is inthe 5-position or 7-position.
 47. A compound of claim 45 in which a is 2and R' is in the 5- and 7-positions.
 48. A compound of claim 47 in whichA is ethylene, a first R' is in the 5-position and is lower alkyl, and asecond R' is in the 7-position and is --N(R¹)₂ or --NO₂.
 49. A compoundof claim 48 in which the first R' is methyl and the second R' is--N(R¹)₂ wherein each R¹ is independently hydrogen or lower alkyl. 50.The compound of claim 49 in which the first R' is methyl and the secondR' is --NH₂, namely 7-amino-2-ethoxy-5-methyl-4H-3,1-benzoxazin-4-one.51. The compound of claim 48 in which the first R' is ethyl and thesecond R' is --NH₂, namely7-amino-2-ethoxy-5-ethyl-4H-3,1-benzoxazin-4-one.
 52. A compound ofclaim 48 in which the first R' is methyl and the second R' is --NO₂,namely 2-ethoxy-5-methyl-7-nitro-4H-3,1-benzoxazin-4-one.
 53. A compoundof claim 48 is which the first R' is ethyl and the second R' is --NO₂,namely 2-ethoxy-5-ethyl-7-nitro-4H-3,1-benzoxazin-4-one.
 54. Apharmaceutical composition which comprises a therapeutically effectiveamount of a compound of claim 13, of a pharmaceutically acceptable acidaddition salt thereof, in admixture with at least one pharmaceuticallyacceptable excipient.
 55. A pharmaceutical composition which comprises atherapeutically effective amount of a compound of claim 27, or apharmaceutically acceptable acid addition salt thereof, in admixturewith at least one pharmaceutically acceptable excipient.
 56. Apharmaceutical composition which comprises a therapeutically effectiveamount of a compound of claim 33, or a pharmaceutically acceptable acidaddition salt thereof, in admixture with at least one pharmaceuticallyacceptable excipient.
 57. A pharmaceutical composition which comprises atherapeutically effective amount of a compound of claim 44, or apharmaceutically acceptable acid addition salt thereof, in admixturewith at least one pharmaceutically acceptable excipient.
 58. A method ofinhibiting serine proteases in humans and animals which method comprisesadministering to a human or animal in need of such treatment a compoundof the formula: ##STR57## wherein: a is an integer of 1 to 4;A is abond, or alkylene having one to eight carbon atoms; R is hydrogen,phenyl, imidazolyl or cycloalkyl having three to six carbon atoms,wherein the phenyl or cycloalkyl ring is optionally substituted with 1-3substituents independently selected from the group consisting of loweralkyl having one to four carbon atoms, lower alkoxy having one to fourcarbon atoms, --N(R¹)₂, --NO₂, halo, and lower thioalkyl having one tofour carbon atoms; and at least one R' is represents a substituent atone of the 5- and 7-positions, wherein said substituent is independentlyselected from the group consisting of lower alkyl having one to sixcarbon atoms, lower alkenyl having two to six carbon atoms, lower alkoxyhaving one to six carbon atoms, --NO₂, halo-lower alkyl having one tosix carbon atoms, lower alkylthio having one to six carbon atoms, halo,--N(R¹)₂, ##STR58## --NR¹ COR², and ##STR59## in which each R¹ isindependently hydrogen or lower alkyl having one to four carbon atoms,or together form a piperidine or piperazine ring optionally substitutedat the ring nitrogen with lower alkyl having one to four carbon atoms or--CH₂ CH₂ OH;each R² is independently lower alkyl having one to fourcarbon atoms, and A is alkylene if R is hydrogen,or a pharmaceuticallyacceptable acid addition salt thereof.
 59. The method of claim 58 inwhich the serine protease is human leukocyte elastase.
 60. The method ofclaim 58 in which the serine protease is trypsin.
 61. The method ofclaim 46 in which a is 1, R' is in the 5-position and is lower alkyl,lower alkenyl, lower alkoxy, halo-lower alkyl, or lower alkylthio. 62.The method of claim 58 in which a is 1, R' is in the 7-position and R'is --NO₂, --N(R¹)₂, ##STR60## --NR¹ COR², or ##STR61##
 63. The method ofclaim 58 in which a is 2, a first R' is in the 5-position and isselected from the group consisting of lower alkyl, lower alkenyl, loweralkoxy, halo-lower alkyl, and lower alkylthio, and a second R' is in the7-position and is selected from the group consisting of --NO₂, --N(R¹)₂,##STR62## --NR¹ COR², and ##STR63##
 64. A compound of the formula##STR64## wherein: a is an integer of 1 to 3;A is a bond, or alkylenehaving one to eight carbon atoms; R is hydrogen, phenyl, imidazolyl orcycloalkyl having three to six carbon atoms, wherein the phenyl orcycloalkyl ring is optionally substituted with 1-3 substituentsindependently selected from the group consisting of lower alkyl havingone to four carbon atoms, lower alkoxy having one to four carbon atoms,--N(R¹)₂, --NO₂, halo, and lower thioalkyl having one to four carbonatoms; R"' is hydrogen or lower alkyl having one to six carbon atoms;and each R' is independently selected from the group consisting of loweralkyl having one to six carbon atoms, lower alkenyl having two to sixcarbon atoms, lower alkoxy having one to six carbon atoms, halo-loweralkyl or lower alkylthio having one to six carbon atoms, halo, --N(R¹)₂,##STR65## --NR¹ COR², and ##STR66## in which each R¹ is independentlyhydrogen or lower alkyl having one to four carbon atoms, or togetherform a piperazine or piperidine ring optionally substituted at the ringnitrogen with lower alkyl having one to four carbon atoms or --CH₂ CH₂OH;each R² is independently lower alkyl having one to four carbon atoms,and A is alkylene if R is hydrogen.